Cloning of pgRNA

 Introduction

This past week in lab, we continued the mission we set out on. We intend to clone pgRNA with kanamycin, to produce a guide plasmid for ours's, and the rest of the lab's endeavors, one that works well with deinococcus. We troubleshoot restriction enzymes and start getting our stock of kanamycin up. We also reattempt pwtCas9 transformation, in preparation for the overarching mutagenesis project that this guide plasmid will fit into. These events went as followed: 

Methods

PWTCas9 Transformation 

1. Perform transformation of D. sonorensis with pwtCas9 plasmid following transformation protocol.

2. After 24-48 hour incubation, inoculate onto plates of both TGY and TGY dosed with ampicillin.

3. Incubate at 30C for 24-48 hours.

4. Observe results and passage successful transformation growth onto TGY plates with ampicillin.

Restriction Enzyme Digest

1. Use XBAI restriction enzyme to digest pgRNA following given restriction enzyme protocols.

2. Incubate at 37C overnight.

3.Run on a gel and look for bands resolving into linear forms.

PCR on Kanamycin Gene Fragment

1. Prep 2 24uL rxn PCR tubes with Q5 master-mix. 

2. Use extracted pRHAM for template.

3. Run through PCR.

4. Confirm products through gel electrophoresis.

Results:

Growth was seen on one transformation plate with TGY and ampicillin and confirmed through a gram stain. This was passaged onto a fresh plate and placed in incubator.

(Figure 1. this image showcases D. sonorensis transformed with pwtcas9 grown on a plate of TGY.)

(Figure 2. showcases D. sonorensis transformed with pwtCas9 grown on TGY dosed with ampicillin. The morphology appears to be that of D. sonorensis, which indicates that this transformation attempt was successful. It's growth on ampicillin entails that D. sonorensis took up the plasmid and is using the AmpR gene to survive the dosage of antibiotic).

Based on how the gel turned out, PCR on the kanamycin fragment was successful. It appeared to be around 900bp.

The XBAI Restriction Digest on pgRNA seemed to be successful. Previously on a gel, the plasmid took multiple forms and showed up as different lengths on a gel. The bands have resolved into the linear form of the plasmid, indicating success with XBAI. 

(Figure 3. The image above displays the gel electrophoresis results of both digested pgRNA and Kana PCR fragments. The pgRNA, in lane 4, has resolved mostly into one, linear band. Lanes 6 and 8 were loaded with kanamycin PCR product, which both consistently show up around the expected 900 bp mark). 

Discussion:

This past week we reattempted the pwtCas9 transformation. Not much was different with this attempt  compared to the last. We continue trying with TGY over R2A to see if added nutrients will increase the potency of the AmpR gene, if integration of the plasmid into D. sonorensis was successful. It seemed that one of the TGY+Amp plates had growth on it, but also had clear contaminant on another side of the plate. A gram stain of what was believed to be sono was taken and looked acceptable. I inoculated this onto a fresh plate of TGY +Amp, with hopes that it grows with no contaminant and we can perform our rounds of confirmation to deem this transformation a success. 

The first time we attempted restriction enzyme digest on pgRNA this week, some mistakes were made. We failed to realize the emphasis of buffer strength to the rest of the recipe in the procedure and also failed to consider the ratio of DNA to enzyme the protocol called for to see any success. Due to this, we added too much buffer, which would destroy any sort of reaction from taking place. From this, we learned that certain ratios must be maintained. For every 10ul of total volume, 1ul should be buffer. Then, for every 1ul of enzyme, 1000ng of DNA is to be added. We redid the tubes following these ratios, which gave us success, as the different forms of the plasmid linearized on our gel. This means that a cut with the XBAI enzyme must have been made. Next week, we plan on double-digesting the pgRNA before running it on a gel to excise. With both of the chosen restriction sites on pgRNA digested with the enzyme, post gel excision, they should be good to go for ligation with the kanamycin fragment.

PCR went very smooth for kanamycin, and now we have a lot of product to work with for cloning attempts. Next week, we will prep the kana fragment for cloning by gel excising and cleaning up this product. More information is to come with this project.





 







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