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!!