Mutagenesis of D. sonorensis: Week Two

 Introduction: 

The previous week in lab involved a series of plasmid extractions to collect the bank of plasmid we will need to complete mutagenesis. From there, we started preparing the materials called for in the protocol we plan on following for cloning in the pgRNA plasmid vector. We started building our primers for the kanamycin gene we plan to target with PCR to clone into our vector. We reattempted transformation of pwtCas9 as well. This blog will go through how we went about the week and our results that follow. 

Methods

1. Grow E. coli with pRHAM on LB plate with kanamycin concentration of 30ug/mL.

2, Grow E. coli with pgRNA on LB plate with Ampicillin concentration of 50ug/mL.

3. Grow E.coli with pwtCas9 on LB plate with Ampicillin concentration of 50ug/mL.

4. After 24-48 hour incubation period, gram stain each plate to confirm bacterial species.

5. Refer to Zyppy Plasmid Miniprep Kit to extract plasmid from each culture after overnight growth.

6. Run plasmid extracts on a gel to confirm results by comparing bp fragment size on gel with known bp size of plasmid.

7. Perform transformation protocol using D. sonorensis cells and pwtCas9 plasmid solution. 

8. After 24-48 hour incubation period, plate transformed cells onto TGY+0.2ug/mL agar plates and onto plain TGY plates as a control group. 

10. Confirm results by observing for growth of transformed cells on antibiotic plates. 

Results:

Nanodrop Results

Plasmid

ng/µL

A260/A280

A260/A230

pRHAM

84.3

1.93

2.10

pgRNA

105.6

1.92

2.14

pwtCas9

162.9

1.93

1.81

(Figure 1. displaying nanodrop readings from plasmid extraction. Purity ratios indicate no DNA contaminants and minimal-no organic contaminants).



(Figure. 2 showcasing plasmid extracts ran on a gel to confirm fragment size. pgRNA size 2600bp, pRHAM size 2300bp, pwtCas9 size 6300bp).

Discussion:

This past week was pretty typical. We prepped all the plasmids, with much stronger yields since switching to the Zyppy kit. With our plasmids ready, we were able to whip up some competent D. sonorensis cells, and get our transformation with pwtCas9 going. This past summer, we had issues confirming transformation with pwtCas9, since the antibiotic resistance biomarkers did not cooperate well with D. sonorensis. AmpR is the biomarker we planned on sticking with, despite its deleterious nature with any species in Deinococcus. In past attempts to confirm transformation, we plated transformed cells onto R2A+0.2ug/mL agar plates, with no growth resulting. We want to find a way to make the antibiotic resistances found on the plasmid work, before being forced to switch cas9 plasmids completely. With this next series of transformations, our hopes are that switching to a media that's richer in nutrients, such as TGY, will give the bacteria more of a fighting chance under the stress of the antibiotic. On the day we performed transformation, I prepared some TGY that we would pour and plate tranformed cells onto, for the following day. Unfortunately, the next day I realized too late that I had forgotten to put agar into the media, leaving us with TGY broth we would be unable to use. With limited options, we just had to plate the transformed cells on TGY plates with Tetracycline and know that the plasmid would not likely be kept. I placed the transformation tubes back into the incubator for the weekend, but realized they should have gone into the fridge to allow us to replate the following week. When we come back into lab next week, we will plan on reperforming the transformation and make the correct TGY. We will also begin gel excision and purification of pgRNA to start the DNA cloning process. A full recap of these events will be covered in the next blog post.


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