Mutagenesis of D. sonorensis; Week 1

 Introduction:

This past summer, the time in lab has been focused around the successful mutagenesis of D. sonorensis. Our preliminary goal is to target the Carotenoid pathway to "turn off" the production of the classic pink pigment, which will result in white D. sonorensis. We began with a proof of concept trial using pRad1, which went smoothly and showed us transformation can be done with D. sonorensis. Since then, our transformation attempts with the plasmids needed for mutagenesis have posed challenges. We have been unable to confirm any successful transformation with the plasmid responsible for cutting the target gene, pwtCas-9. To confirm transformation in the cell, any particular plasmid will contain antibiotic biomarkers that will gift the cell a new resistance to the specific antibiotic on the given plasmid. The biomarkers found on pwtCas-9 do not cooperate well with the bacteria chosen to transform. The TetR gene has been a poor biomarker, since D. sonorensis itself already has strong, inherent Tetracycline resistance, so zero distinctions can be made from a transformed vs typical d. sono cell, as they both can grow overnight on strong tetracycline concentrations. The other biomarker found on pwtCas9 is AmpR, which has not yet been useful, since ampicillin at nearly any concentration will obliterate the D. sonorensis cells, even when resistance is added through the plasmid. 

One other challenge, which will be a main focus at the start of this semester, will be preparing the guide RNA plasmid to target the intended gene, and to provide correct biomarkers that work with our species of bacteria. This blog post will take you through our initial steps taken to overcome these challenges. 

Methods:

1. Prepare plates of LB media with added antibiotic based off what plasmid is found from each bank of bacteria. (Ex. E.coli with pRad1 needs to be grown onto LB medium with ampicillin or chloramphenicol)

2. Grow bank of E.coli with plasmid by inoculating onto it's respective antibiotic plate: pRHAM E.coli grown on kanamycin, pwtCas9 E. coli grown on ampicillin, pgRNA E. coli grown on ampicillin. Incubate at 37C 24-48 hours.

3. With growth on plates, perform plasmid extraction protocol on pRHAM, pwtCas9, and pgRNA. (Refer to Zyppy plasmid miniprep kit protocol for details). 

4. Confirm results with gel electrophoresis 

Results: 

Fig. 1- Nanodrop from Plasmid Extraction 8/23

Plasmid	ng/uL	260/280	260/230
pgRNA	78.0	1.87	2.22
pRHAM	46.1	1.92	2.09
pwtCas9	58.4	1.85	1.72

(Figure one. displaying the amount in ng/uL of plasmid extracted from E. coli and its purity ratios, indicating samples free of organic and nucleic contaminants). 

Fig. 2- Gel Electrophoresis 

(Figure two showing bands at the correct size for each plasmid)

Discussion: 

This past week was full of plasmid extractions. We had to reperform the protocol multiple times since our nanodrop purity ratios were low, indicating we either didn't lyse the cell properly to have enough DNA to work with, or we had some sort of organic contamination. Our first attempts were made using the Thermofisher GeneJET Plasmid Isolation Kit, which seemed to be the source of our troubles. We do not typically use this kit, but had to out of necessity. After our first batch of plasmids with the kit turned out poorly, we paid closer attention to each step of the procedure to see what may be going wrong. Typically, the start of the protocol involved a lysis step, where a buffer is added that breaks apart the cell wall, followed by a neutralization step, which returns contents back to preferential conditions. These two steps form a large bank of precipitate at the bottom of the tube, composed of the cell wall and all cellular contents from inside, due to proper lysis. The formation of this precipitate is critical in the protocol, since cells must be open in order to collect it's DNA. With the GeneJET plasmid kits we used, only one of the three plasmids being extracted were lysed and formed precipitate. We moved  forward in the protocol with the lysed tube, which was pgRNA. We still had poor results on the nanodrop, upon which we were advised to stick to the Zyppy plasmid prep kit solely. This change made all the difference in our quality of extraction, and yielded the results as seen above. Another note I made when working with this kit is to use smaller pipettes on the part of the protocol where the cellular contents are centrifuged to a pellet, and you are instructed to remove the supernatant (containing plasmid DNA) into a separate column. Using smaller pipettes decreases the likelihood of the pipette tip interacting with the pellet directly, or being accidentally sucked in with the supernatant due to a wider tip's greater pipetting pressure force. 

After the success of our plasmid extraction, it became time to confirm these results by double checking that the band size of the extracted fragment matches up to the known bp size of the plasmid. This is done through electrophoresis, where a 1% Agarose gel is made to load samples in. Our first attempt to load the gel with our samples came with an array of issues. We had previously prepped the gel and stored it for later use, but little did we know, we used water as the solvent instead of the TAE buffer that is critical in allowing electricity to run through the sample. We would come to find this out after first troubleshooting with the samples. When preparing a sample to be put onto a gel, the ratio of dye to sample must be retained in order for the dye to bind to the DNA correctly and give the best results. For each well, we prepared 5ul water, 2ul dye, and 5ul sample. This should come out to 12ul of content for each well, but when we went to pipette everything, we had volume left over. Since our ratio was now off, we had to go in and add more dye and sample, which gave us a total volume of 15ul. When working with such minute volume, any small deviation can impact what volume one may think they're working with. It's important to pay close attention to the stopper of the pipette, and never push past the stoppers resistance, or you will pull a larger volume than the pipette is set to. When we loaded the gel and hooked it up to the machine, we set the electricity settings to run at a pace that we expected to take around an hour. Instead, our gel ran through instantaneously, which is when we realized water may had been used over TAE. The next time we entered lab we remade the gel correctly and loaded the samples keeping the previous notes in mind, and didn't come across any issues. Looking at the gel, each band that lit up was parallel to the band size of it's plasmid. Next week in lab, we plan to transform pwtCas9 and grow on TGY with amp to give more nutrients for D. sonorensis to work with and survive the small concentration of ampicillin and keep the plasmid. We will also begin preliminary work to prepare for the engineering of pgRNA and pRHAM.