Constructing Agriculture on Mars: A Study of Azospirillum Inoculation
Advisor: Ms. Gabriella Baessa
Internship Location: GenSpace and Cold Spring Harbor Laboratory
Onsite Mentor: Dr. David Jackson, Casey Lardner, and Dr. Chris Aston
“Turning the red planet green,” has long been an aspiration of modern research. But with the recent development of aerospace technology, supporting the human colonization of Mars may not be as far-fetched as initially thought. Following the successful retrieval of Martian soil and analysis of its composition, the University of Central Florida has synthesized replicant “simulant” soils (MGS-1) equally safe for education and lab-based experiments alike. However, the lack of a proper macro/micro-nutrient profile in Martian soil is a major factor in inhibiting efficient plant growth for hypothetical agricultural harvest. Delivering the required nitrogen compounds to infertile soil has proven possible for the bacteria Azospirillum Brasilense in terrestrial environments, but what about these newly synthesized simulants? This project examines the potential of Azospirillum treatment as a medium of biofertilization for MGS-1, using common alfalfa (Medicago sativa) as a model organism. Through research at the Jackson Lab of Cold Spring Harbor Laboratory and community biolab Genspace, collected data will determine the viability of applying current phytoremediation technology to Martian soil in wet-lab environments. By conducting an analysis of plant growth genes and biomass of greenhouse-grown sample groups, the project will quantitatively compare the plant yields of fertilized Martian soil and agriculture-grade Earth soil. Additionally, by engineering fluorescent strains of Azospirillum, this project will include a thorough visualization and explanation of the mechanisms behind nitrogen fixation within our soil and root systems. The prospect of renewable bacterial fertilizers has vital implications to future efforts in establishing life amongst the extremely low-resourced climate of Mars. This project will hopefully establish a link between nitrogen-fixing bacteria and increased crop harvests on Martian soil simulants.
Project Blog
Week 0: Introduction
Hello all, and welcome to my Senior Project blog! My name is Yuna Stechert, and I’ve been going to BASIS Independent Brooklyn for ten years. I plan on studying biological engineering in college, and have been interested in plant physiology and incubator construction since seventh grade. Previously, I’ve explored botanical science with the community biolab Genspace, including a minor involvement in microbiology. Utilizing my previous experience, I intend to grow the model organism Alfalfa (or Medicago Sativa) using customized incubators with different types of soil (both terrestrial and Martian). Then, I will work with my off-site mentor Mrs. Baessa to treat the plant groups with a common plant growth promoting bacteria– Azospirillum Brasilense. The goal of this project is to demonstrate that Azospirillum Brasilense works as a fertilizer when used on replicant Martian soil, similarly to earth-tested methods. For my on-site internship, I am tentatively considering studying at the Jackson Lab at Cold Spring Harbor Laboratory. But In the next ten weeks, I will outline my research process in and out of the wet-lab environment, and deliver a scientific paper summarizing my findings. Thank you for reading, and I look forward to updating soon!
Week 1: Lab Tour
Welcome back to my blog, and thank you for joining me!
In this first week, I confirmed my placement at the Cold Spring Harbor Laboratory (CSHL) and focused on constructing the layout for plant groups that will grow during my internship.
As the Lab itself contains up to nine plant-growth environments, including greenhouses, CO2 chambers, and incubators, selecting the ideal conditions for alfalfa growth took priority before germinating our seeds. Therefore, early on in the week, I coordinated with Prof. Dave Jackson of the Jackson Lab to predict possible formations for growing Martian, terrestrial, and project control groups.
According to previous agricultural studies, alfalfa grows most efficiently at approximately 70 degrees Fahrenheit (or room temperature). Since the CO2 chambers and incubators were needed to accommodate plants of more fragile temperature ranges, Prof. Jackson agreed to place the alfalfa in CSHL’s outdoor greenhouses. Because the goal of this project is to determine the viability of bacteria fertilizers on Martian soil, the results are dependent on comparisons between my alfalfa’s survival in Martian and Earth soil; this meant that I had to organize the flats in a way that would accurately address each factor I was concerned about.
I decided to test my hypothesis using four factors: the presence of alfalfa, ammonium nitrate, Azospirillum bacteria, and type of soil (Mars/Earth). After establishing at least two positive and negative controls each, and four total experimental groups, my flat prototype was prepared and compiled into Figure 2. The chart will be referenced during the final plant-growth trials in April.
Following my remote research, I traveled to Cold Spring Harbor in Long Island to tour the lab and plant informal testing groups. The Jackson Lab specializes primarily in plant biology and genetic engineering, so I learned about some of their research that increased the seed/kernel yields of corn from ~16 rows to 20-24 rows. Prof. Jackson and my assigned Lab mentor Matias Gleason also familiarized me with the resources around the facility, including growth chambers and lab desks (Figures 3 & 4). Finally, I planted trial seeds in 4 flats (Figure 5) using the two different soil types of the lab, Arabidopsis and Maize, in order to determine the Earth soil that we will use for final tests. The trays were laid in Greenhouses B & A and will continue to be monitored by greenhouse staff (Figure 1).
After spring break, I hope to test concentrations of Azospirillum bacteria on the plants and shadow Matias in his PhD research labs. See you next week!
Week 2: Transgenic Plants & Greenhouse Update
Hello, welcome back to my blog!
I was off to an early start this week, traveling again to the CSH Lab on Tuesday following some minor preparation during spring break. After arriving at 10 am, I rode up towards the greenhouses to meet Matias (my project supervisor) and plan for a fruitful day of research.
CSHL had everything from fish hatcheries to GMO, bunching space tomatoes, so it was extremely exciting to explore the equipment and staff that produced such a wide range of specialization. At the Jackson Lab, most experimental work is dedicated to plant genetics and agriculture. Therefore, I spent the day shadowing his PhD research into the other model organism Arabidopsis (Figure 1), which is typically used because it demonstrates high visibility into genetic mutation– meaning that whenever Arabidopsis plants are mutated, they tend to show outward signs (phenotypic variations), allowing scientists to more easily draw links between different genes and their relationships within the plants circuitry. To summarize his research, Matias is focused on proving the existence of mRNA (genetic material that instructs the cell to produce specific proteins) movement across cells in Arabidopsis. To do so he is constructing transgenic plants using agrobacterium (a bacteria that can introduce foreign genetic material by infecting host plants) in order to capture visual proof of mRNA movement between cells.
As my individual project focuses heavily on the expression of plant growth genes, and the interaction between bacteria and plant growth environments, Matias was a highly fit as a mentor due to the similar circumstances of our research. So allow me to share a few highlights of the on-site work:
First, I was able to tour a more controlled growth chamber within one of the agriculture houses. Matias kindly showed me around the facilities including some of his current plant groups (Figure 2). I helped weed and maintain some of them, which will later be used in various genetic crosses to provide evidence for his thesis. Seeing the more sterile environment of growth prompted my curiosity and possibly exposed some flaws in my experimental design. I did initially want to ensure a good degree of sterility, which isn't as likely within the outdoor greenhouses that we planted groups in last week. Ultimately I decided to research more within the next week to determine ideal conditions (temperature, humidity) for growth, and make a decision whether or not to plant future alfalfa in the growth chamber instead. I then left for a lunch break, accompanied by the furry friend of a professor at the lab (Figure 3).
Later on, using previously incubated (grown) Agrobacterium, we performed a “flower dip” procedure. The agrobacterium (Figure 4) was isolated and purified by a series of solutions and centrifuge runs, then added to the plants to infect them by submerging the flower stalks and reproductive tissue within the culture (Figure 5). This process was especially exciting because I had worked with agrobacterium before at the Genspace Open Plant community project, but not in such a direct procedure. We discussed more in depth about our research, including a plasmid (form of DNA) for agrobacterium that Matias has been developing that works via the root system of plants. In future weeks, I may work with him to transform some of the Alfalfa for my project using this plasmid in order to detect differences in plant growth between transgenic plants and non-GMO ones in the MGS-1 stimulants.
I was able to gain a lot of experience with transforming plants this week, so I’m looking forward to returning next Thursday with more insight from Mrs. Baessa and Genspace members.
To conclude the week, I spoke with Mrs. Baessa, who ordered a few more supplies to return with, including: more kilograms of MGS-1, freeze dried Azospirillum to rehydrate for experimentation, and pH/nitrate testing strips for chemical analysis of our soils. Next week I hope to create an in depth conditions sheet for my alfalfa, and start informal groups to grow in the Martian soil stimulants.
I look forward to updating you all with my next lines of research!
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Weeks 3-4: Final Trial Preparations
Hi! Welcome to the third and fourth weeks of my project blog!
The past two weeks have been primarily remote because my project advisor (Matias) was traveling for a scientific conference. On the bright side, Mrs. Baessa and I had plenty of supplies available to work with, and extra time at Genspace allowed me to gain some insight into a possible agrobacterium transformation for alfalfa.
After visiting CSHL the past week, I determined that testing the two soil groups prior to planting would be ideal. With nitrogen compound testing strips, I tested the available MGS-1 simulant for its nitrate and nitrite concentrations. As predicted by the simulant spec sheet, nitrate/nitrite levels in the soil were shown as zero (Figure 1). So in the following growth trial, soil will be collected and subsequently tested to determine the amount of nitrogenous compounds produced by the alfalfa root system, Azospirillum fertilizer, or both.
Additionally, previous meetings have drawn my attention to creating a conditions sheet for alfalfa in order to determine the best environment at CSHL for running the final trial next week. My research was rooted primarily in humidity and temperature, because the laboratory separates several chambers based on their sterility, humidity, and temperature. Meanwhile other factors such as soil depth, pH, level, and watering may be adjusted accordingly by the users preferences. Ultimately I determined that experimental groups will require full sun, humidity of approximately 60-70%, temperatures of 60-65 degrees Fahrenheit, and a randomized block layout to grow most efficiently given the circumstances of my research. A randomized block design refers to a system of distributing experiment groups completely randomly within a given space in order to reduce result biases due to uneven conditions; given that the greenhouse/growth chambers that I will use may have varied conditions across the different flats I use, next week I will randomize the placement of several hundred plant trays.
Finally, to end the week I visited Genspace to continue working with the Open Plant Community project. With several other members, I transplanted common liverwort (also known by its scientific name Marchantia) from agar plates to larger boxes for long term storage (Figure 2). Our propagations included several strains of Marchantia, namely Tak1, Cam1, and RUBY - a red variety cultivated by inserting a plasmid that expresses the red pigment betalain (commonly observed in beets). While alfalfa and liverwort only share the title of “model organism,” agrobacterium affects them equally. I hope to pursue producing transgenic cultures of alfalfa with Matias’s plasmids in the coming weeks, and look forward to documenting differences between Marchantia Polymorpha and M. Sativa transformation.
See you then!
Week 5: Plants!
Hey y’all! So glad you could join me for the fifth week of my blog!
Firstly, I am extremely grateful to announce that Matias and I were able to sow the final test group of alfalfa plants on Thursday, April 25th. In the last blog, I outlined my process of brainstorming variables to control for this final group; my focus for this week shifted towards implementing these controls in the greenhouse environment.
It is the perfect time for alfalfa growth right now! Temperatures in Cold Spring Harbor are becoming more agreeable to both plants and people, while the photoperiod (ratio of light to darkness time each day as sensed by crops) indicates to many seeds that it is the ideal season for germination. These factors encouraged my decision to plant this week, as well as the fact that I will have around 40-50 days of growing time before collecting biomass samples (and more sets of data!) for my final presentation.
Earlier in the week, I prepared a few other updates to my experimental design that I’d love to share. To predict the approximate size of the roots that will grow in the final trial, I uprooted two plants from Week 1 to see if we could downsize our area of the potting inserts. The initial trays were around six square inches– which I downsized to two based on the larger plants (Figure 1). Additionally, my plan to use a random block design from Weeks 3 & 4 came to fruition as I discussed what a reasonable number of experimental replicates for each treatment would be with Matias.
The least amount of replicates recommended by Matias was three, and as such, I decided to include four pots per group without alfalfa, and eight pots per group with an alfalfa plant. But before randomizing the positions of each potting insert, I had to consider a constraint of the greenhouse flats: treatment groups could not be mixed in the same flat.
As visualized in Figure 2, Each flat comes with a solid tray at the bottom, followed by a drainage tray, and then finally inserts at the top. According to Prof. Jackson, any treatments applied to the plants that grow in a given flat will migrate across the solid tray, contaminating all groups on the flat. Because of this, I had to randomize the layout of this experiment categorized by treatment type: None (Negative Control), Azospirillum Brasilense (Biofertilizer, Experimental), and Ammonium Nitrate (Chemical Fertilizer, Positive Control). These groups were organized and numbered to be sown. (Figures 3 & 4)
After deciding on the design layout, we organized and built the greenhouse flats to completion, filling the various groups according to their intended soil-type, plant amount, and fertilizer-type. Appropriate safety equipment was used during the planting, as the Martian soil simulants are extremely fine and possibly irritating, but opening up the astro-soils was an incredibly rich experience. We watered each group after adding the soil, and the simulants were highly sand-like, mimicking the dusty, but dense nature of non-domesticated growing environments like Mars. (Figure 5)
What Next?
In the next few weeks, I plan to inoculate (treat with bacteria) the reserved Azospirillum flats. For the sake of accuracy, Matias and I will verify the CFU (colony forming units) of the Azospirillum Brasilense projected by its spec sheet. CFU are a good measure of the amount of bacterial colonies contained by a given freeze-dried bacteria after rehydration. We intend to grow a collection of ~100 CFU, which will be counted after being cultured in a LB (Lysogeny Broth - A medium of nutrient-rich agar used for growing bacteria) plate *without antibiotics.
Regarding research, I will be assessing whether Azospirillum Brasilense can be “marked” (attached to) a fluorescent protein that will make it glow under imaging software. If the bacteria responds well historically to GFP (green fluorescent protein) or RFP (red fluorescent protein), then we may be able to visualize the movement of the bacteria around the plant's root system for the final project. On another note, I will be reading a few papers from CSHL postdoc Penelope Lindsay, who is well-versed in the relationships between bacteria, fungi, and plants.
Thanks for tuning in, and I look forward to updating as our plants grow!
Week 6: Solutions, Equations, and Germination
Hi-ya! Welcome back to the sixth blog of my Senior Project!
To recap, last week I was able to fill and sow my main alfalfa groups, which will be stored in the greenhouse until they reach maturity (~40-50 days). These plants and the biomass samples that can be collected from them will serve as the exhibit for many experiments to evaluate their overall health in the future. Lots of hands-on lab work going on this week, so I’ll jump straight into the important stuff.
Firstly, our plants got their initial dose of fertilizer this week! Thursday began promptly with checkups around the greenhouse, a routine I plan to keep constant throughout the remainder of this project. When designing my experimental plan, I had ensured that the plants had at least one week to adjust to the greenhouse environment, after which I would begin to inoculate the inserts of the Azospirillum labeled groups. Inoculation (in this instance) refers to the process of introducing a species of bacteria to a given environment, and we decided to perform it “in-furrow,” meaning the soil around each seed was treated with a bacterial solution. Other methods include spraying the plant, dipping the seeds, dipping the roots, and many more.
Fertilization
In anticipation of the bacterial treatments, I calculated several variables we needed to prepare an appropriate concentration for the individual inserts. The recommended doses for Azospirillum began at 1 Gallon per every 100 ft2, a metric appropriately scaled to handle large agricultural plots. As each of my inserts have an area of 4.15 square inches, I converted the concentration to a more agreeable unit: 260 μL (microliters) per in2. The powdered Azospirillum (Figure 1) that we bought comes in an inert carrier, which holds 10^7 CFU/Gallon; CFU is “colony forming units,” indicating the number of bacterial colonies that will grow in a given amount of solution. To calculate the required volume we needed to fertilize each plot, I multiplied the 260 μL concentration by the area, yielding a dose of 1079 μL/insert.
With a total of 24 inserts requiring our biofertilizer, Matias and I prepared a solution with 75mg/50mL (the same concentration calculated according to the recommended treatment). Azospirillum powder was weighed via precision scale, and mixed in solution with distilled water. (Figure 2)
After preparing the materials for inoculation (Figure 3 - drawing), we drove up to the greenhouses to assess and treat the plant groups from last week. I was happy to see that plants were successfully germinating in the control and earth experimental groups (Figure 4).
Greenhouse Update
On a more depressing note– MGS1-planted seeds, however, were lagging behind the earth groups and did not germinate (boo!). A scary sight for sure, but not at all unpredictable due to the ruthless composition of the simulant. I hypothesize that this stunt in growth was likely due to the density of the soil: when prodded with a glove, the surface was solidified, mimicking a compact feel, like concrete. Optimistically the problem is a lack of water retention by the Martian simulants. MGS-1 possesses a more sand-like quality that causes water to evaporate. Alfalfa seeds, or any seed for that matter, require a baseline humidity in order to break the dormant phase they naturally fall into. Plant germination requires several factors, like sunlight, even temperatures, but also water –something evidently absent from the cake-y textures of the Martian soil groups, but abundant in each earth tray.
This setback for germination in my experimental plants was certainly alarming, but I am staying optimistic that the plants also lack the required macronutrient Nitrogen (and its derivatives). Inoculating with Azospirillum is designed to introduce the missing nitrogenous compounds and stimulate plant growth responses. So with the previously prepared bacteria solution, I micropipetted 1079 μL of treatment into the non-control and negative control groups.
After finishing inoculation, we left instructions for the greenhouse staff to keep the Martian groups wet throughout the growth cycle. They will bottom-water the flats every 2 (Mars) to 3 (Earth) days.
Finally, As mentioned last week– I also prepared a solution of A. Brasilense to incubate in LB agar. After taking a 10 mg measurement of the bacteria and creating a stock solution in 1000 μL, a serial dilution was performed, and 1 μL of the stock was added to 99 μL. The resulting solution was 100 μL to be streaked (spread evenly across) an agar plate without antibiotics (Figure 5). The test was performed twice across 2 plates (total 200 μL) and set to incubate at 37°C for a week.
What’s next?
With the final project approaching in a month and a half, next week I will be discussing a literature review of bacteria movement visualization techniques. Our intention is to develop a protein marking system to compile imagery from the activity of Azospirillum as it colonizes the roots of alfalfa– this will be integral in describing the importance of a healthy root system, and showing how nitrogen fixers interact with the native microbiome of plants underground.
See you next week, ciao!
Week 7: Microscopy, LB, and Wall-E
Hi plant people! Thanks for joining me for the seventh week of my project update!
Speed recap:
Last time at the lab, we treated the greenhouse flats with and without alfalfa by inoculating them using a measured bacterial solution. Additionally, our Mars plants were observed to be lagging behind their terrestrial counterparts in germination time; and finally, I prepared a plate to grow an Azospirillum colony test separately, which I will count by hand this week.
The timeline for our project was conveniently time-generous this week, so my focus shifted primarily to researching plausible concepts for use with the aforementioned transgenic bacteria (referenced briefly, Week 2). Before diving into the wet-lab work, I’d like to introduce some relevant background from bacterial genetic engineering.
Agrobacterium:
Agrobacterium tumefaciens (not to be confused with my biofertilizer, Azospirillum) is a bacteria you might frequently encounter in nature. While you might not readily recognize it, if you’ve ever seen a tree, bush, or other foliage brandishing an absurd, tumor-looking protrusion on its tissue, you’ve seen Agrobacterium! The microorganism species is a common soil-dwelling microbe that introduces its genetic material through a circular DNA structure called a plasmid (Figure 1.1). These plasmids (specifically described as “Ti Plasmids,” or Tumor Inducing Plasmids, for Agrobacterium) have been a holy grail for plant genetic modification; their natural makeup includes a segment referred to as “transfer DNA,” or T-DNA, that is extracted from the plasmid to infect the host plant nuclei (Figure 1.2). Infection of the plant’s genome is initiated through the release of phenolic compounds (derived/adjacent to phenol, C 6H 5OH), a common stress-signal response, which alert virulence genes to facilitate movement of the tumorigenic T-DNA into the recovering plant.
So… why is Agrobacterium so important for genetic engineering?
Although its typical composition breeds a cancer-like phenotype, the tumor-producing genes within the T-DNA sequence are only a portion of the bacterial genome. They are encapsulated within the plasmid by a left and right border group, which essentially tell the organism to insert everything in between their limits. So when removing the tumorigenic genes, and releasing the left and right borders intact, researchers are left with a mechanism to infect plants without the integration of the tumor growths.
Empty T-DNA are commonly referred to as “disarmed,” and can be programmed by geneticists with replacement genes– any sequence chosen to take the place of the Ti characteristic. These transformed Agrobacterium plasmids can be taken and used to infect a range of host plants with irregular phenotypes, a tool useful for research, agricultural advantages, and medicine (Figure 1.1).
This process is the elected method of Open Plant for their insulin-expressing liverworts, and also of many Cold Spring Harbor PhD students when proving the existence of various biological processes.
Matias is using Agrobacterium to create many mutant plants for his project, so in the next few weeks we will be working with one of his transgenic plasmid constructs to add a fluorescent protein (known as m-scarlet) into the A. Brasilense fertilizer colonies. The ultimate goal is to express both a glowing red pigment and antibiotic resistance in a new generation of bacteria, where the pigment will allow us to document the root colonization during fertilization, and the resistance will act as insurance we can verify the successful implementation of foreign genes. After transforming the Azospirillum, we will grow them on antibiotic agar plates, and the surviving colonies will evidently contain the fluorescent and antibiotic resistance traits, as they will be linked together on the plasmid! (Meanwhile colonies without said genes will not survive due to the presence of an antibiotic, which naturally kills Azospirillum and Agrobacterium.)
Fluorescence Microscopy
Now, I have an interesting preview for all of you! In the lab this week, Matias already had some samples of his model (Arabidopsis Th., Figure 2) marked with two types of fluorescent proteins: RFP (red fl. protein) and GFP (green fl. protein). These leaf segments were assayed under the CSHL fluorescent microscope, which excites the proteins within the tissue, giving visibility to the movement of cells they are contained in (Figure 3).
LB Plate Colonies
On another note, last week’s Azospirillum plates have finished incubating as of this Thursday! Counting both trials and taking an average of their colonies, I ended up with ~500 colonies instead of 100 (Figure 4). This was perplexing, but not at all a setback in my research, as I’ve documented now that our bacteria are more concentrated than initially thought. However, it was certainly exhilarating to see the emergence of colonies after working with seemingly inorganic powder for so long!
Greenhouse Update:
Enough bacteria talk… Now to the greenhouse progress, where I’m excited to announce that we have Mars growth! After surveying the Earth groups per usual, I detected a small but vigorous sprout shooting up from the Martian simulant insert #16 (Figure 5). What great news! A single biofertilizer-treated seed has sprouted, with hopefully more to follow. If all goes well, we will have happily abundant harvests from the Martian plot in a few weeks. Our sweet ecosystem seems to be adjusting well to the moisture change from last week, and I look forward to updating in stride.
What's Next?
Along with more greenhouse updates, the upcoming week will include more specifics of our Azospirillum plasmid design, and how Matias and I will adapt the current Agrobacterium strand. Additionally, I intend to sterilize a group of alfalfa seeds so that they can germinate without interference from the MGS-1 simulants, the hope is that root development in a less strained environment will breed a higher rate of post-germination survival.
Cheers!
Week 8: Gases, Fun, and Azospirillum
Greetings, salutations! Welcome to the eighth week of my blog, and thanks for joining me once again.
Recap:
Here’s the ritual breakdown– Last week, we discussed the importance of the soil-dweller Agrobacterium tumefaciens, and the mechanism it uses to transfer its DNA to host organisms. As for hands-on experimental work, I counted some Azospirillum colonies after rehydrating them with distilled water, and discovered a Martian-grown alfalfa sprout, huzzah!
Cold Spring Harbor was beautiful as ever this week, and I’m noticing more and more fruit and flora emerging as spring reaches full throttle. There were even wild strawberry blossoms lining the walkway to Delbruck Lab (The plant research lab at CSHL); life inside was equally active. (Figure 1)
To start off the week, I designed a plan to germinate some alfalfa plants without interference from the harsh regolith (loose rock and dust) environment. The reasoning behind this was to document a visual difference between Azospirillum-inoculated root development and control seeds without the fertilizer; this way, once the alfalfa sprouts are transplanted into their respective soil environments, their early development may provide them with a stronger chance at life.
Chlorine Gas Sterilization:
When picking up the typical household cleaning agent, the fine print of many products may include an important lesson in chemical mixology: always read the safety data sheet. Disclaimers on your average bottle of toilet or tile cleaner will urge against the layering of cleaners and disinfectants for good reason! This is because however efficient it may seem, combining the latent chemical profiles of agents like bleach, cleaners, or dissolvables can form a dangerous cocktail of products– so it's with great caution that I caution– do not try this at home!
Bleach, also known as Sodium hypochlorite (NaClO), when combined with the compound Hydrogen Chloride (HCl), a common ingredient in many cleaning agents, can form motile chlorine gas (Figure 2). Chlorine gas is highly toxic due to its reactivity as a halogen. Diatomic chlorine gas molecules attack the bonds of water molecules, forming free oxygen as well as HCl, a potent acid able to severely deform cells in the body.
However, while chlorine gas is deadly to people in long-term exposures, it holds quite the advantage in lab sterilization protocols. The reaction between Chlorine and water contains intermediates that penetrate a negatively charged cell membrane in microorganisms, causing premature cell death and reproductive failure in these cells. Because of its efficient disinfecting properties, chlorine gas is used as a sterilizing instrument in many laboratories.
At CSHL, as well as a majority of plant and microbiology laboratories, a popular medium of growing and storing organisms is agar, a seaweed-based solidifying agent that can be supplemented with nutrients or deterrents of many kinds. Its dexterity is a double edged sword, however, because storing a plant without necessary precautions may breed fungus, mold, and bacteria– all which pose risks to the intended organisms longevity. (Figure 3)
Therefore, chlorine gas is a great way to eliminate foreign contaminants when plate-ing seeds, sprouts, and many forms of plants. And it applied favorably to our alfalfa seeds! So on Tuesday, I ran the procedure as follows:
Alfalfa seeds were added to open microfuge tubes and strapped to the edges of a gas chamber. Then, in the fume hood, a beaker containing 50 mL of distilled water, and 50 mL of standard bleach was placed within the center. And to create the Cl gas, 6 mL of HCl is quickly added to the beaker. As the gas forms quickly, I placed the chamber lid on quickly before allowing the gas to sit for 1 - 1.5 hours with the seeds. (Figure 4 - video)
Following the seed sterilization, Cl gasses were evacuated from the chamber and allowed to dissipate, while the neutral product left is an electrolyte solution of table salt (NaCl) and water.
Agar Germination:
Disinfected seed groups were plated on MS (Murashige and Skoog - a powdered blend of nutrients beneficial for supporting agar plant growth) supplemented agar, using a micropipette and sterile water. (Figure 5). Next week, these plate-germinated seeds will be carefully transplanted into a new plot of Martian soil tests in order to weigh the benefits of isolated germination.
Plasmid Mapping:
On another note, if you are interested in genetic mutations, engineering, or bacteria, I’m thrilled to introduce the key components of our transgenic (containing foreign genetic material) Azospirillum this week! My previous blog articulated the basics of plasmids and genetic transformation, which I will be utilizing to build fluorescent versions of our nitrogen fixing bacteria.
As Azospirillum brasilense does not naturally contain the fluorescence phenotype, this is a factor that must be introduced via genetic alteration. With Matias’s help, I will be combining two of his previously stored plasmid builds into a form receivable by our model bacteria.
To build some background, promoters are sequences of DNA that instruct the cell where (and of what quantity) to send proteins after they are translated from RNA. These sequences are foundational to building sets of DNA because isolating genetic material from one organism requires an agreeable promoter in order to be successfully expressed in another. Allow me to explain:
For example, the gene that encodes for our fluorescent protein of interest is referred to as “mScarlet,” originally sampled and sequenced from a species of coral. Once tissues containing these genes are collected and purified, researchers will have access to the sequence. These lines will contain first the coral-specific promoter, and downstream the gene itself (Figure 6).
However, in the instance that someone (for example, me) wanted to express that same gene within a bacteria (for example, Azospirillum), the coral-specific promoter would be largely unfit when attached to the mScarlet gene. Because there are vast anatomical differences between corals and bacteria, if a coral-promoter-containing plasmid was added into bacteria, the promoter would essentially tell the cells “send a lot of this protein to the polyps and tentacles,” to virtually no effect, because bacteria do not have polyps and tentacles. This short circuit imposes the need for geneticists to split the sequence and discard (or save for later) the original promoter, and add a promoter fit to the model organism.
This is what we are doing! MScarlet is our exact protein of interest, providing the brightest red fluorescent pigment to date! And in the coming weeks, I will be inserting the promoter from a bacterial plasmid (Figure 7, right) into a construct containing the saturated FP (fluorescent protein - Figure 7, left).
The bacterial promoter we intend to extract is from the plasmid created in Gabriele Labes’s “Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn1696 as reporter,” and is referred to as the pc promoter.
Greenhouse update:
Back to the plants!--
Once again, it’s time for bacterial treatments. Following the procedure outlined in Week 6, I added the same stock solution of Azospirillum to replenish the bacteria soil groups.
As for growth checks, the earth control groups are demonstrating clear differences in both height and biomass production when at different levels of biofertilizer. In Figure 8, the non-treated Earth group (left) is much smaller than the treated Earth group (right). These controls can help demonstrate the effectiveness of our Azospirillum for improved vigor in the final research article.
Unfortunately, no new sprouts have emerged from the MGS-1 simulants (Figure 9). The single stalk from Week 7 is still going strong, but if the lack of germination persists I intend to outsource my experimental design in order to produce a better sample size for the final biomass weigh-in and tests.
What's Next?
My next line of action includes planting the germinated seed groups in our MGS-1 regolith, and Chlorine-sterilizing another group of seeds to keep for colonizing with our fluorescent Azospirillum. Further, we will begin modifying the MScarlet plasmid to be compatible with our bacteria, and a stock solution of electrocompetent (ooh!… what's this!..) cells will be frozen for DNA insertion.
Thanks for joining me, and stay tuned!
Week 9: Codons and Cloning
Hey blog! It’s nice to see you, welcome to the trials and tribulations of week 9– I’ve got lots to share today!
Previously, we sterilized and plated a few groups of alfalfa seeds for a germination test. Additional to those, I worked with Matias to select and plan a genetic map of our fluorescent Azospirillum colonies, which will be constructed in the following weeks. Finally, we re-fertilized all of our greenhouse groups, plus a complete survey of their germination rates and vitality.
Germination vitality assessment:
Root health and soil compatibility pose great significance to the vigor and long-term survival of plants. Because of the coarse and dry nature of MGS-1, I anticipated previously that it would be difficult to trigger germination in our alfalfa with only a natural germination schedule. Therefore, in addition to sowing seeds straight into each soil group, a supplementary trial of agar-germinated plants was conducted last week– and oh they’ve grown! (Figure 1) The control group (left) showed promising root growth, only mildly overshadowed by the Azospirillum-inoculated ones (right).
Since we are living one week post-plate, this is the time to plant them. In a group of twelve, the visually strongest seedlings of Azospirillum were transplanted into a new plot of MGS-1, mimicking the insert size and configuration of the regular experimental trial. Next to that, nine non-inoculated sprouts were transplanted into equal conditions in more MGS-1 (Figure 2).
As for our Greenhouse Check…
Considering the modifications to climate (increasing humidity, frequency of watering), the soil-germination groups planted back in Week 5 are looking unfortunately desolate. The main MGS - Azospirillum group appeared largely dried out and cakey, despite our numerous attempts to work with these harsh conditions. As such, our singular prized plant has unfortunately dried out in the fourth week, weighing an approximate 0.6 mg. This was a loss for sure, but I’m staying optimistic about my research thus far– many signs have shown that while Azospirillum did create enough nutrients for germination, long term growth requires fundamental changes to the soil’s chemical composition.
Additionally, my experimental plan is largely reliant on our trials yielding detectable amounts of plant harvests, especially an average biomass comparison across our different conditions. As such, the limited growth is posing increasing risk towards a conclusion for my paper– thus I’ve made the decision to consult alternative sources in order to mitigate the setbacks created by MGS-1’s severity.
In my literature review this week, Karthik Chinannan’s (et. al) “Effects of Mars Global Simulant (MGS-1) on Growth and Physiology of Sweet Potato: A Space Model Plant,” drew my attention to a plausible path to success. By supplementing the simulant with different concentrations of earth soil, they avoided necrosis and germination failures, and were able to analyze several components of the resulting crops’ health. The paper, published in 2024, is a promising lens for Martian agriculture; thus, I will be adapting my trials to its likeness next week in order to remediate the harsh chemical profile of these simulants.
Colonial Microscopy preparations:
On a more exciting note, practical preparation for our fluorescent bacteria (RFPB) figures has begun! Managing this process included several tasks, including:
Germinating groups of alfalfa to be inoculated by the RFPB (below)
Creating Electrocompetent Azospirillum colonies (details later)
Editing MScarlet to include a start codon (details later)
Combination of MScarlet and bacterial promoter into final plasmid (next week)
To start, we needed to create another germination plate of regular alfalfa. Contrary to the agar plates last week, these sprouts will not be transferred into soil; instead, they’ll be incubated together with our fluorescent bacteria colonies once developed, and captured via a fluorescent microscope.
This time, four tubes of selected seeds were sterilized using another Chlorine gas reaction, and the MS-media plate procedure from Week 8 was repeated for two of those tubes. The remaining two tubes were stored in a cold room– they will be plated in equal conditions next week.
Building Electrocompetent cells:
This component of the RFPB figure is largely about preparing Azospirillum to receive foreign genes (the plasmid we are developing, in this case). Here’s what I mean:
The Azospirillum we have right now are completely natural, containing their standard genome without any alterations. What we want to accomplish is the addition of foreign DNA fragments like MScarlet and a bacterial promoter, but their current state won’t allow the intake of substitutional genetic material because they are entirely stable with a natural genome.
So what researchers have done in the past is developing a cell state called “competence,” or willingness to incorporate external DNA into the cellular genetic makeup. Competence is ideal for transforming Azospirillum (and many other microbial species) because in stressful conditions, bacterial DNA will become damaged and responses will prompt the intake of floating plasmids or fragments outside of the cell membrane (Figure 3).
Our chosen method uses electroporation, the process of shocking cells with an electric pulse to produce competence. So, beginning with a culture of Azospirillum, we took periodic scans of their absorbency, the amount of light absorbed when a certain wavelength is fired through a sample of material. Absorbency has an inverse relationship with concentration, because more light absorbed through a sample means less material blocking the reception of light, and a smaller concentration.
Checking the absorbance is important because electroporation is ideal when a bacteria sample reaches the key “log-phase” of their growth state; this is when the rate of multiplication is the highest (Figure 4). Thus, waiting until their absorbance reached ~0.5 OD (Optimal Density), yielded the Azospirillum we needed– which were immediately put on ice to halt growth.
Next, cells were pelleted, meaning spun on 4,000 to 14,000 revolutions per minute in a centrifuge until collected in a clump, and suspended in a mixture of ice cold water and glycerol. These solutions were stored overnight after being frozen with liquid nitrogen (Figure 5).
On Friday, we ran the electroporation procedure, pulsing the cells in a cuvette with ~1700 Volts. Immediately after inducing competence, the Azospirillum was transferred to a liquid nutrient solution (LB - Lysogeny Broth), and refrozen.
Start Codon Addition:
And for the final RFPB update– the first stage of transformation took place this week!
Here's some background: Our template DNA (the one we are going to add fragments to) is MScarlet, a red fluorescent protein in plasmid Matias’s stock; however, it's currently missing an integral component of all genetic translation mechanisms, the start codon.
When proteins are synthesized from DNA, the process occurs in two steps: transcription and translation. First, an enzyme known as RNA polymerase binds to DNA, and transcribes the sequence into a complementary mRNA molecule– the form readable by ribosomes within a cell. Perceptive ribosomes are then able to compile a series of amino acids into a chain (also known as a protein) based on the information provided by mRNA, aided by a supplementary tRNA that brings miscellaneous amino acids from the cell to be turned into proteins, this is known as translation.
Any instance of translation in the cell begins with the start codon, AUG, or methionine, which signals to the ribosome where to begin synthesizing a protein.
As mentioned before, sourcing the MScarlet plasmid from Matias’s collection came with its own complications, and we need to add a start codon to it in order to produce a viable fluorescent protein code. Otherwise, the MScarlet proteins (the aspect that glows) won’t be produced from the fragment missing its start codon.
So to conclude, we ran a PCR (Polymerase Chain Reaction) to introduce methionine upstream of our MScarlet fragment. Two primers were used to amplify– primers being small fragments of DNA that bind to a template vector, hanging off by several bases that encode for additional genes. Using start-codon specific primers, we mixed the template plasmid, two primers (forward and reverse), and a Master Mix (A stock solution of many A, T, G, and C bases in suspension, as well as the enzyme Taq Polymerase). The role of Mastermix is for the Taq enzyme to use the abundance of free bases and skillfully attach them according to the primer sequence. These bases extend the template strand. (Figure)
The PCR was set to run 34 cycles in the temperatures 95 °C, 62 °C, and 72 °C at differing intervals, then stored at 12 °C. These will be tested in E. Coli cultures next week!
Whats Next?
Soon, I intend to implement the new greenhouse strategy, supplementing MGS-1 with earth soil in order to create a set of new germination plots. Further, we will introduce our new start codon MScarlet line to a culture of E. Coli, culturing them on antibiotic plates in order to verify successful transformation. If successful, these cells will be melted again and the plasmid isolated for storage!
Thanks for reading, looking forward towards Week 10!
Week 10: Martian Mixology, Molecular Biology
Hiya! Happy Week 10, and welcome back to the blog!
Summary:
Here’s your recap: Following the arrival of our selected plasmids, we added a missing start codon in front of the MScarlet vector; the finished products (labeled Mscar-start) were stored to be entered into an E. coli test this week. Also, we started to germinate more seeds that will be colonized by the finished fluorescent Azospirillum, the goal is to show how our bacteria move (via the color of the fluorescent proteins) when interacting with the alfalfa underground. Finally, we shocked a culture of cells into the competent state, which will be thawed and combined with the finished transgenic plasmid at the end of the project.
Space Travel, Farm Expansion:
This week we focused largely on the remediation of our Martian soil groups. My previous literature review presented a captivating pathway to long-term survival, contrasting our current standing with only a few sprouts and their biomass (or lack thereof). According to the paper, when faced by sparse germination rates in Martian simulants, researchers were able to mitigate key issues like the dry texture and small particle size by introducing concentrations of Earth soil. Although you may be saying, “duh, plants grow in Earth soil,” I’d like to propose a line of thinking that occurred to me when confronting the issue of transporting terrestrial growth mediums to Mars as opposed to our original plan of only seeds, Azospirillum bacteria, and the materials to multiply it on arrival:
1: All plants create waste of some kind, even the most efficient, manicured sweet potato (a crop that continuously produces tubers) will drop leaves as the plant grows older. This fact allows for farm expansion if waste from their life cycles are repurposed into a soil-fixing supplement.
2: Given the opportunity to spread farmland as harvests are made, the initial amount of soil can be limited to a small area.
Sustaining one person on a vegetarian diet required approximately 0.32 acres of farmland; if we supplemented 25% of that volume with Earth soil, however, we’d have to carry 160,000 lbs of it on the ship with us (14x the weight of the spacecraft itself, and only for one passenger.) This is utterly impractical and we’d sooner just farm hydroponically; but if harvest waste is considered, the issue of transport becomes much more transparent.
The natural recycling process that occurs as plants shed layers opens the pathway to reduce that soil volume to as little as 54 lbs (although this is just a calculation, and would need to be simulated), as each crop produced will directly fuel the colonization of more Martian soil plots (Figure 1).
This plan is rather optimistic, but given that water is equally or more scarce as food during space travel, utilizing the life cycle of plants to our advantage proves itself as a necessary means for survival.
Considering this idea, for the purposes of thoroughly studying the viability of Azospirillum biofertilizer on Martian soil, I supplemented Earth soil with a concentration range of 0%, 50%, and 75% MGS-1. This alteration was fashioned due to the unforgiving dryness of 100% MGS-1 as noted in previous blogs, and will still allow us to study the effects of our bacteria on growing plants while (hopefully) exponentially stimulating their germination rates.
New Growth:
During the preparation of these new Earth-supplemented growth trays, I maintained the control groups that we previously outlined, expanding them to encompass the different combinations. They were as follows:
Group type Concentrations Treatment type, Purpose
Negative control: 0% MGS-1, 100% Earth–None, establish typical plant growth standard
Experimental: 0% MGS-1, 100% Earth–A. Brasilense, identify standard fertilizer effect
Negative control: 50% MGS-1, 50% Earth–None, establish baseline Martian plant growth
Positive control: 50% MGS-1, 50% Earth–Am. Nitrate, establish growth standard with successful Nitrogen implementation
Experimental: 50% MGS-1, 50% Earth–A. Brasilense, test biofertilizer on 50% MGS-1
Negative control: 75% MGS-1, 25% Earth–None, establish baseline Martian plant growth
Positive control: 75% MGS-1, 25% Earth–Am. Nitrate, establish growth standard with successful Nitrogen implementation
Experimental: 75% MGS-1, 25% Earth–A. Brasilense, test biofertilizer on 75% MGS-1
These groups are intentionally designed to analyze how Azospirillum compares to standard plant growth, and chemical alternatives.
Each insert was prepared with the outlined volumes of each soil, and sowed with Medicago sativa (Figure 2). Greenhouse updates will return routinely next week as I check these tests!
Moving on, I’d like to draw your attention to the progression of our bacteria-Mscarlet protein engineered plasmids! As I mentioned last week, we ran a procedure to add the missing start codon component to the Mscarlet gene fragment. This week, E. coli were inoculated with that plasmid.
E. coli are an ideal candidate for testing for transformation success because they are extremely fast growing, while Azospirillum typically takes around three days based on our experience. So how do we verify that the ATG start codon was introduced and integrated into our bacteria?
There are many ways to do it, but our chosen method is to grow colonies of the E. coli on antibiotic treated plates. Why? Because the start codon we attached also encoded a gene for antibiotic resistance, specifically the compound Chloramphenicol (“CHL”). So, in plating these microorganisms in a resistant media, survivors will be limited to the ones containing a start codon-MScarlet plasmid. This process is called “selection,” similar to natural selection in evolution, but artificially constructed to study bacterial traits.
After growing overnight, our plate was fully saturated with successful groups (Figure 3), Hooray! Now that we understood the state of the plasmids, our goal was to separate that genetic material from the E. coli; after all, our goal is to add them to Azospirillum in the end.
DNA isolation and extraction:
To extract the start-Mscarlet plasmids, we began by spinning (or what's called “pelleting”) the E. coli down for 12,000 revolutions in one minute around the centrifuge. This process formed an opaque pellet of bacteria at the bottom of my tube, allowing for the empty solution to be discarded. (Figure 4, example)
Next, a suspension buffer was added to the bacterial clump in order to both re-liquify the culture, and dilute this next reagent– Lysis buffer; lysis (greek for “loosen”) is a disruptive solution that functions to dissolve the cell membrane and release genetic material inside. During this process, cells are killed, but if gently washed away, DNA remains intact and usable after the lysis is removed. So, our lysis buffer was added after the suspension buffer, and I gently inverted the tube and centrifuged the solution for another 12,000 revolutions.
After, we add the next step of DNA extraction: “stop N3,” an opposite of lysis that counteracts its ability to damage the cells, and precipitates (solidifies) the dead cell capsules (Figure 5). The dissolved bacteria received the N3, and were then spun at 13,000 revolutions per minute to spin out the dead cell gunk.
Finally, the leftover solution was added gently to a collection tube (Figure 6) in order to wash out the remaining discards and isolate the DNA completely. Taking a wash buffer, an additional liquid that dissolves cell matter, but plasmids are insoluble in, we rinsed out the tube. Then, DNA was collected using distilled H2O, which flushes out the water-soluble circular DNA.
In my humble opinion, science is security; this is because a vast majority of biology is entirely invisible to the human eye, therefore many steps must be taken to enlarge results to verify what we think is happening is actually happening. This is where the nanodrop comes into play!
Through shining a light across samples of liquid, the nanodrop is able to determine concentrations of proteins, DNA, and RNA within them. Ours at CSHL takes a single microlitre of solution, and is overseen by a small minion attachment, very cute! This machine processed our E. coli-isolated plasmid, and pumped out the following image (Figure 7 & 8).
As you can see, the largest curve was present around 260 nanometers, this is wonderful! DNA is detected across that specific range, meaning that the reading of 122.1 nanograms per microliter can be assumed as our plasmid.
SP Videography:
So much genetic talk… I’d like to take a moment to share a definite highlight of my week at the lab now!
On Thursday, I had the genuine pleasure of working with Mrs. Jo Goldfarb (of BASIS Brooklyn) and Mr. Troy House to direct a tour of CSHL. Jo and Troy were the most incredible company, and Matias and I were able to participate in the filming of a fun project to be coming out after the Senior Projects! As I look forward to sharing these inside features, here is a preview of what’s to come: (Figure 9)
What's Next?
Approaching the next week, my goal will be to treat the newly sowed groups with their needed treatments, aka A. Brasilense and Ammonium Nitrate. In the wet lab, we will take the isolated plasmids and begin to introduce a bacterial promoter, completing our fluorescent prototype. These genetic components will also be going through a “digest,” and “ligation,” (more on that later).
That's all for now, see you all next week!
Week 11: Agents and Acquisition
Hey plant people, thanks for joining me once again for Week 11!
Summary
Last time, I covered the bases of altering our greenhouse trial design to include different amounts of Earth soil. These changes were designed to allow for greater germination rates so that we can ultimately compare the plant growth across the concentration range of MGS-1. Other than the plant stuff, engineering our fluorescent Azospirillum has been going fairly well, with our E. coli test coming back positive for the start codon we added.
After verifying our success with the MScar-start template, we began a lengthy, but productive process of work of adding the bacterial promoter region for the final construct.
If you’re unfamiliar, I covered the relevance of each component of the plasmid in Week 8. But this week, we worked in three steps to finish the transgenic material: Addition, Digestion, and Ligation.
Addition of the Bacterial Promoter:
As mentioned previously, the promoter region of a genetic template is what encodes the amount of proteins produced by a cell, and where they are sent. By connecting a bacterial promoter, we are able to alter the fluorescent, coral derived-MScarlet plasmid to work in bacterial species (such as Azospirillum).
Thus, on Tuesday we thawed the MScarlet, start codon template strand as preparation for a PCR (Polymerase Chain Reaction).
Because the PCR system recommends 10-30 nanograms of DNA for plasmid preparation, we diluted our original 122.1 ng/uL DNA solution 10 fold (9 uL ddH2O : 1 uL plasmid), yielding a product of approximately 12 nanograms of material per uL.
2 uL of that dilution were added for a total of (12 + 12) 24 nanograms of DNA! **Here’s a small tip, it is typically good practice to add the largest volume of reagent when performing procedures of such small volumes. So note that before the 2 uL of template plasmid were added, we added 25 uL of MasterMix stock and 19 uL of distilled water. Then, we had to add 2 uL of PCR primers.
Primers are the incredibly important part of a PCR that determine what the final product will include. For some background, a PCR (Polymerase Chain Reaction), works by rapidly running DNA through a preset temperature cycle. This temperature cycle includes three important steps, and they go as follows (Figure 1):
Denaturation: double stranded template DNA is heated until unwound into two single stranded sequences.
Annealing: At a cooler temperature (specifics vary depending on the fragment), primers are allowed to “anneal,” or attach to the single stranded templates.
Extension: Utilizing floating base pairs within the MasterMix stock, a DNA Polymerase enzyme synthesizes a new strand of DNA starting from the primer. The bases are bonded through the enzyme onto the single strand, forming an end product of new DNA.
After the entire PCR, genetic material is amplified as the steps repeat until the concentration has increased exponentially.
Now, the primers that researchers select determine the location that DNA Polymerase begins to form the new DNA. This means that adding the primer that both encodes for your gene of interest, and connects correctly to the base strand is key. To do so, Matias and I worked using SnapGene, a sequence simulation providing insight into the plasmids of our project.
PCR primer design is a delicate art. You have to include what’s called a “GC clamp,” a Guanine-Cytosine pair at each end of the sequence; this is because GC base pairs contain triple bonds as opposed to the Adenine-Thymine double bond of weaker strength. The clamp ensures that your primers are strong enough as to not come apart at their ends during the denaturation phase. Further, at least 50% GC content is needed throughout the entirety of your primer, to prevent any splitting around the rest of the strand.
Simulations like SnapGene (a free alternative is benchling.com, figure 3) allow researchers to pursue this specific purpose, displaying the GC content, melting temperature, and other relevant details for highlighted sequences of DNA. So, in utilizing SnapGene and Matias’s guidance, I designed the two primers (Forward-FW and reverse-RV) needed to attach the bacterial promoter (labeled “bac” for brevity) to the MScar-start template. These are the two that we settled on and ordered for the week:
FW: aagcctgttcggttcgtaaactGGGTCTCGTATGGTGAGCAAGG (52.2% GC)
RV: tacgaaccgaacaggcttatgtCAAGGTGTCACCACCCTGCC (54.7% GC)
**lowercase letters include where the primer will be synthesized, while UPPERCASE letters are the region that anneals to the template strand.
They arrived at the lab before our transformation procedure, so we proceeded accordingly. Each primer comes in a dehydrated form, and must be both rehydrated and diluted. Distilled, nuclease free water was added to each tube, and spun down via centrifuge at 12,000 rpm. The resulting product was mixed with a micropipette, with 10 uL taken out and diluted by a 90 uL amount of distilled water.
After preparing the primer solutions, 2 uL of the FW and RV were added to the PCR mixture mentioned above; the resulting solution was 50 uL that we soon added to a PCR thermocycler. (Figure)
Gel Electrophoresis:
Post PCR, we verified the length of the plasmid through Gel electrophoresis– a method of measuring DNA based on its mobility relative to electric charges (Figure 4).
Here are the basics:
Given a sample of DNA like ours, we take around 5 uL of plasmid solution and mix it with a loading dye, weighing it down as well as staining it to be visible. In an electrophoresis tank filled with buffer solution, the mixture is inserted into wells along with a ladder (reference material containing many different lengths of DNA).
Because DNA possesses a negative charge, placing small amounts onto a porous material like agar (and here's the key) with positive and negative electrodes active allows the DNA to move away from the negative electrode and towards the positive (opposites attract). This mobility is dependent on the length of each fragment; larger strands of DNA (ex. 10,000 bases) will move less as they are heavier relative to their smaller parts (ex. 500 bases).
Our intended plasmid with all of its parts (start codon, bacterial promoter, and MScarlet genes) should be approximately 3,000 base pairs in size. And so, measuring it post-charge against the appropriate DNA ladder allowed us to verify its completion (Figure 5).
Success! Our PCRs both matched clearly at the 3 kilobase mark, with the first result looking slightly more clear. (**note: I ran two PCR tubes because I’m a novice, and one might have failed during the many steps; more experienced molecular biologists would likely only need one)
Digestion of Foreign Contents:
Welcome to step two! After measuring the DNA with our gel, I decided to take an additional step for security– the Plasmid digest.
Digests have one goal: remove all foreign contents/fragments from a sample of DNA (the start-bac-MScar in our case). To do so, we gently mixed it in solution with an Exnase enzyme. Exnase is quite an interesting tool for genetic engineering! Roughly, it disassembles natural genetic material based upon their elements, but completely ignores the presence of synthetic DNA, eliminating the waste products from transgenic manufacturing.
During this process, ~500 nanograms of plasmid were added (3.5 uL of 122.1 ng/uL solution) to a mixture of distilled water (14 uL), buffer solution (3 uL), and Exnase enzyme (0.5 uL). A temperature cycle was used to store, activate, and control the enzymatic reaction, this took place in another PCR machine. (Figure 6)
Ligation of Linear DNA:
We’re on the final stretch! I previously explained how plasmids are circular structures of DNA that are present in many bacteria, however, when PCR is performed, the denaturation step disrupts the loop of DNA, turning it into a linear sequence. Although our PCR products are now successfully 3 kilobases, and waste-free, they still must be turned into a form with that circular shape. This process is called Ligation. (Figure 7)
Ligation occurs through the use of another enzyme, ligase; it alters lines of DNA by joining two complementary ends together and forming our desired structure.
In our ligation, a recommended 2.5 uL of plasmid were added into distilled water (20 uL), a buffer solution (4 uL), and ligase enzyme (2 uL).
Finally, the circular, digested, bac-MScar-start plasmid was completed and stored frozen.
Greenhouse: Treatments
Now for the plant stuff… opening last week with a new trial of plants means that this week is treatment time! This time around we have two solutions of interest: the A. Brasilense and (new!) chemical control Ammonium Nitrate (Figure 8).
Azospirillum Brasilense was prepared at the previous concentration of 75 mg in a 50 mL solution, which is approximately 3,000 colonies per plant insert. To cover the full 4.15 square inches of each insert, 1079 uL of bacteria was pipetted around each plant.
Ammonium Nitrate was weighed in at 1195 mg and suspended in 25 mL of distilled water. This solution is accurate to the 224 kg/ha scale recommended by agriculture experts. 1,000 uL of the nitrate solution was pipetted into each insert.
Greenhouse: Plants:
As for the progress in growth, the earth supplementation seemed to have worked to increase germination rates! This was a major relief after struggling with the soil’s unforgiving chemistry for so many weeks; the plants grew the lowest in MGS-1 without any treatment (Both 50% and 75% groups), and the highest in our A. Brasilense 50% MGS-1 (Figure 9). Ammonium Nitrate 50% MGS-1 groups followed shortly after.
Although these are just the first week of plants, I’m ecstatic to watch their progress!
That concludes an eventful 11th blog… thanks so much for reading and I look forward to updating next week, Ciao!
Week 12: Green Thumb
Hi everyone, welcome to week 12!
Summary
Here's your recap: Previously, we completed the construction of our bacterial, start codon-containing MScarlet plasmid after performing a PCR gene addition, waste digestion, and DNA ligation. Also, in the greenhouses, our alfalfa sprouts were developing nicely after the transition from 100% MGS-1 to 75% and 50%. The plants were treated according to our experimental plants with A. Brasilense and Ammonium Nitrate as well.
However melancholy, we are nearing the end of this project; as such, this week was dedicated to compiling results from our greenhouse trials, and building the final presentation for Friday. As I mentioned previously, the three categories by which we are evaluating are as follows:
Nitrate/Nitrite soil concentrations– through nitrogenous compound detecting strips.
Chlorophyll Uptake– through a chlorophyll absorbance meter.
Total biomass– through weighing wet mass on a precision scale.
These assays were conducted through the processes below.
Results: Nitrogenous compounds
To compare the amount of nitrogen introduced by our biofertilizer against normal levels of Earth/Martian soils (and the chemical competitor Ammonium Nitrate) we began by collecting soil samples from each group (Figure 1). 50 milligrams of regolith mixture were measured from each sample, then suspended by 20 mL of distilled water; water-soluble nitrates are able to escape the soils upon immersion in this solution, allowing us to measure the nitrogen content of each experimental group through test strips (Figure 2). After submerging the papers for approximately 2-3 seconds each, they were labeled and dried to be compared against the nitrate gradient guide.
The final results were certainly promising (Figure 3), with the Azospirillum treated groups demonstrating substantially higher nitrate content compared to the initial 0 ppm we measured previously. The highest Nitrate concentration was group (H) the 75% MGS-1 with Azospirillum, while the lowest levels were the same across all non-treated groups.
Results: Chlorophyll Absorbance
Moving on, we also measured the level of absorbance of our plants so far. Using an atLeaf CHL STD meter, chlorophyll content in each leaf was calculated (Figure 4). These machines allow us to detect the pigment concentration in a given sample using a programmed wavelength of light; the beam is shot through a leaf and detected by a meter behind it. Absorbance across each sample holds an inverse relationship with the amount of chlorophyll in the leaf, because pigment functions to intake the light and convert it into energy; more pigment of the plant means less light detected through it.
With the atLeaf value detected, we converted the chlorophyll content into mg/cm^2, compiling our data into the following figures: (Figure 5)
As shown, the chlorophyll averages across our experimental groups followed their Nitrogen content quite similarly. Plants primarily utilize reactive nitrogen to produce chlorophyll and facilitate photosynthesis, shown by the roughly 70% of uptaken nitrates/nitrites that are accumulated in their leaves. Because of this, our chlorophyll assay demonstrates the correlation between nitrate levels in the soil, and efficient uptake and usage of these resources by the plants we grew.
Notably, upward trends were noted as treatments were added, with the exception of Earth with Azospirillum. This may be erroneous work on my part, because the plants might have actually overgrown to the point of yellowing from lack of space in their pots (Figure 6). The leaf yellowing of this specific group contains comparatively less chlorophyll, but may not represent the uptake efficiency as they were green last week and had just overgrown their small inserts.
Otherwise positively, Azospirillum was able to outperform both our plain and chemically fertilized soils in both 75% and 50% MGS-1. In the 75% MGS-1, without treatment, causing plants to germinate proved highly difficult (hence the 0% chlorophyll from 0 plants grown) meaning that only with the addition of exogenous nitrogen (Nitrate/Nitrite/biofertilizer) were they successfully able to grow. This data was also mentioned in the biomass weigh-ins, outlined below.
Results: Biomass
Now for the final assay! Considering that the biomass of our crops is the factor that influences actual functionality (because it determines how much is harvested and eaten hypothetically in space), this is what I consider the most important result of our project. So, after taking all of the relevant details for the last 2 tests, plants were uprooted and washed with distilled water to remove excess soil (Figure 7)
To measure the amount of biomass produced in each experimental plot, I dried off these plants and weighed them on our lab-grade precision scale (Figure 8).
The values of each plants weight were recorded in the graph below:
As outlined, the relative biomass of each group demonstrated thoroughly the ability of Azospirillum to increase the plant growth; Granted, the effectiveness of A. Brasilense and Ammonium Nitrate did decrease as the concentration of MGS-1 increased from 50%-->75%, but it facilitated both germination and amplification of crop mass.
All of the results considered, I was utterly relieved to receive confirmation of the fertilizer’s success. However, we did have a failure this week…
Fluorescent Bacteria:
Unfortunately, after testing the completed plasmid in E. coli, isolating the successful groups, and growing colonies of Azospirillum with the new transgenic material, the phenotypes of our bacteria failed to demonstrate the traits for fluorescence. Although this molecular cloning portion of our project was a side project meant to visualize below-the-soil Azospirillum colonization, I was nonetheless distraught by this result. Matias and I checked each plate thoroughly, but no colonies were glowing red despite their notable resistance to the antibiotic placed on our agar media. But I’d like to introduce a key observation during the final steps of this experiment: the E. coli tests did glow. (Figure 10)
This fact alone confirms that our engineered plasmid does function successfully to introduce the MScarlet gene to bacteria, but our error likely lies in the method with which we introduced the genetic material to Azospirillum. Although Azospirillum is a common nitrogen fixer, it was difficult to find a complete method to introduce this specific gene; as such, with little time before the presentation, we simply had to electroporate the cells with test voltages of 2.0 and 2.4 volts. Matias hypothesized that this was likely too weak or too strong for causing the cells to intake our plasmid, meaning that these values may have disrupted the plasmid's ability to intercept the natural makeup of A. Brasilense, yielding colonies without our desired trait. So, although we were not able to produce a glowing colony of Azospirillum, the plasmid that we engineered for the last few weeks does have the ability to transform more common bacteria such as E. coli; our Azospirillum likely requires a bit more tinkering to find out a successful transformation procedure.
Reflecting on this portion of my work, engineering the glowing bacteria would have been incredible, but molecular cloning is a lengthy process that is typically characterized by a lot of trial and error. I am extremely grateful that we were even able to produce a successful transgenic plasmid, and the pursuit of our product is an endeavor that I intend to continue even after the end of this project.
Final Thoughts:
That leaves me to conclusions–
Ultimately, I presented my process and results to a wonderful audience on Friday, June 14th. My slides are shown below, and while this performance concludes my official duties for these assignments, anyone and everyone is welcome to ask questions after the fact!
Constructing Agriculture on Mars: A Study of Azospirillum Inoculation
This senior project has been an absolute joy, privilege, and learning period to experience– I’d like to give my sincerest thanks to everyone who has supported me throughout this process, including but not limited to my faculty advisor Mrs. Gabriella Baessa, my CSHL mentors Matias Gleason and Prof. David Jackson, as well as the larger BASIS faculty for designing the framework and evaluative portion of this time I (and hopefully you) will not soon forget. Once again, thanks for giving me the opportunity to share my research with you! Go Bears!!