Mutagenesis of D. sonorensis; Week 3

 Introduction:

The previous week in lab, our group worked on getting a good bank of our plasmids going, as we start beginning the process of mutagenesis. This past week, we started work with the restriction enzymes chosen based off the restriction sites of the plasmid, where we intend to insert KanaycinR gene to allow pgRNA to work as a viable guide-RNA for not only our group, but for those in the lab working towards their own mutagenesis endeavors. The ligation protocol called for gel excised and purified fragments to be prepared before the ligation steps. This blog post will go through how this was done, and what changes were made to yield more success. 

Methods

Gel Excision and Purification

1.Load sample into gel. Chose a volume with large molecular mass to yield most product through the excision and cleanup process. Expect around half of product to be lost.  

2. Locate target band and complete process referring to Thermo Scientific GeneJet Gel Extraction Kit.

3. Purify sample referring to Thermo Scientific GeneJet Gel Extraction Kit.

4.  Chose an elution volume with consideration of how much DNA is currently present in the frit and an understanding of what the extract will be used for in the future. 

5. Use nanodrop reading to determine if further DNA cleanup kit is needed. 

Restriction Enzyme 

1. Refer to BAM restriction enzyme protocol .

2. Leave in 37C incubator overnight.

3. Run digested DNA on gel to see results. 

Additional Mentions

1. Inoculated more pgRNA E.coli onto more LB amp plates.

2. Inoculating transformation tubes onto test plates. "Transformed with PWTCas9" onto 1 TGY plate and 1 TGY+Amp plate. "Transformed without PWTCas9" onto 1 TGY plate and 1 TGY+Amp plate

3. Another round of plasmid extraction using Zyppy Plasmid Miniprep Kit.

Results:

After the gel excision protocol, it is necessary to observe the yield of DNA on the nanodrop. The purity ratios were nowhere where they should be. The yield was very low as well.  

pgRNA extractions performed at the start of this week were ran on a gel. Results are as follows:


(Figure 1. Gel electrophoresis results of pgRNA plasmid extraction. The various bands are forms of the plasmid. The plasmid can take a supercoiled form, appearing larger in size. Nicked forms can appear on the gel as well.)

pgRNA digested with BAM restriction enzyme was ran on a gel. The different forms of the plasmid seemed to have linearized from the cut made by the enzyme at the site and resolved to one band. 

The transformation of D. sonorensis with pdCas9 was unsuccessful, as the transformed tubes did not grow on the TGY + Ampicillin plates. 

Discussion

The gel excision was not much of a surprise to our group to have yielded the results it did. Since the agarose is a contaminant on the nanodrop, despite the efforts of the gel cleanup, its very likely the remnants of the agarose prevented these ratios from being what we would expect. The yield of DNA was not high, despite loading nearly all of the plasmid we had extracted onto the gel. Throughout the steps of the gel excision and purification, it's was made known that half of the DNA is typically lost. With this in mind, one can imagine how increasingly difficult it may be to yield any DNA from this process with how we had to chose which band to excise for the protocol from the various different forms the plasmid took. The plasmid appeared in the gel in different organizations, ranging from supercoiled, linear, and nicked formations. Since not all the DNA was located in one area, a decision had to be made of which form of band was best to excise. We chose the band with the most visible molecular mass based on the vibrancy of the fluorescent dye. Despite this, one could see almost half of the DNA loaded into the gel remained in the other band variants post excision. This is not as efficient as we would like and is what we think may have impacted the low yield seen. To resolve this, we took a restriction enzyme to one of the restriction sites on the extracted pgRNA. This effectively makes a "cut" in the DNA, which will resolve the various forms of plasmid seen on a gel, into one, linear band. This will be beneficial to our gel excision process, as more DNA will be found in one place, making the band that is cut for the purification steps contain a higher molecular mass. The next week in lab we will reattempt with these changes and see if any beneficial results come from it. 

Our PWTCas9 transformation was not successful for us this week. We initially plated them when we had minimal growth in the transformation tubes, then reattempted the next day with more growth seen. Both of these inoculations did not show any growth from the transformed tube on TGY+AMP. D. sonorensis is intolerant to Ampicillin. This is likely the reason we have not been able to verify a conclusion of these transformations. We tried to dose a more nutrient dense media with ampicillin this time(TGY), but we see this has not seemed to have helped. We do not want to have to tediously clone PWTCas9 with another antibiotic resistance biomarker, so we are trying to navigate our options and hope for the best. The only other biomarker on PWTCas9 is Tetracycline, which doesn't serve any use to use since D. sonorensis has inherent tetracycline resistance.  

Next week, we will reattempt gel excision with digested pgRNA now linearized. We will use pRHAM and run it through PCR to get a large stock of Kanamycin fragment to use to clone into pgRNA. More is to come from this project.

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