Template Plasmid Part 2: Proof-Of-Concept Experiment 1

Hello! Today, we will continue discussing the template plasmid, as introduced in the previous blog post. To recap, template plasmids are the correct form of the mutated target sequence that the DNA repair mechanisms of human cells use as a reference to replace the mutated DNA with the canonical DNA sequence. This week, I conducted research on two aspects: the formation of the template plasmid based on MLH1 and MSH2 and my first proof-of-concept experiment testing whether accurate cloning took place in cells. 

Template Plasmid Formation 

To form a template plasmid based on the specifications of this experiment, a plasmid backbone is necessary. This backbone contains important elements such as restriction enzyme cut sites, antibiotic resistance protein sites, and also gene sequences encoding for different parts of a viral vector. This led me to choose the psPAX2 plasmid backbone as it contains the important elements encoding the IDLV viral vector (gag, pro, pol) and confers ampicillin resistance. To incorporate the template as part of the plasmid, I designed PCR primers flanking the 5’ and 3’ ends of the MLH1 and MSH2 template sequence. These 20-nucleotide primers are then combined with SacI restriction enzyme sites at both the 5’ and 3’ ends of the forward and reverse primers, as seen below. 

MLH1_HDR_Forward: 5’-AAAAAAGAGCTCCTGCCTGGCTAATTTTGTATT-3’  MLH1_HDR_Reverse: 5’-AAAAAAGAGCTCAGAAGAGAATAGATTTTAATC-3’ MSH2_HDR_Forward: 5’-AAAAAAGAGCTCGAAAGATTTGACCATACTGA-3’ MSH2_HDR_Reverse: 5’-AAAAAAGAGCTCTCCCTTGAAGATAGAAATGT-3’    

red → adenine bases for stability    

blue → SacI restriction enzyme overhangs 

These primers are used to insert the template sequence into the plasmid backbone. The template sequence will be cloned from the genome of donor human peripheral blood mononuclear cells through PCR as well. 

Incorporation and Defect Testing 

As mentioned earlier, the template sequence will be incorporated into the plasmid backbone. This incorporation will involve the following steps. First, SacI restriction enzymes will digest both the PCR product (template sequence) and psPAX2 plasmid backbone. Then, the PCR product and psPAX2 plasmid backbone will be ligated in 1:10 molar ratio using DNA ligase. 

The end product of this procedure will result in the formation of the template plasmid required as seen in the figure below:  

Figure 1: Template Plasmid after incorporation steps are complete 

To ensure that the plasmid has no defects, it will be transformed into competent E. coli. To find out whether the transformation worked correctly, E. coli will be plated onto agar plates and mixed with ampicillin, an antibiotic. The DNA from the bacteria that survive after adding ampicillin will be purified by miniprep.  

Cloning Confirmation  

This template plasmid will be run through an experiment to prove whether it contains the template or not. To do this, the psPAX2 plasmid backbone without the template and the psPAX2 plasmid backbone with the template will be digested by two restriction enzymes: Bsu36I (6301) and NheI (7714). These two enzymes were chosen on the basis that they don’t repeat in the empty plasmid backbone and that they will not initiate cuts within the template itself. The two plasmids will undergo gel electrophoresis (using a 1% agarose gel) to find out the length of the fragments formed by restriction enzyme cuts. I predict that the psPAX2 plasmid backbone with the template will have a longer fragment of 1.9 kb compared with the psPAX2 plasmid backbone without the template at 1.4 kb. The results of this can be seen in the figure below: 

 Figure 2: Expected gel results with psPAX2-backbone and psPAX2-HDRtemplate following digestion by Bsu36I (6301) and NheI (7714).