Spring 2024 - Week 1: January 22 - January 26, 2024

 Deinococcus radiodurans Carotenoid Knockout

Introduction: 

Prior to winter break, I was given the opportunity to work on a new project that was separate from my previous work with minimum inhibitory control on different Deinococcus species. While I am still working on that project, my primary focus has shifted to working with a new group to transform Deinococcus radiodurans from a red color to a white color. We hope to achieve this by knocking out the crtB gene in D. rad. While this is only the tip of the iceberg, if we are able to successfully do this, we can go on to transform Deinococcus caeni. 

In order to be able to transform D. rad, we would need to extract separate pieces from different bacteria. We started off by growing E.coli pRAD1 from freezebac and D. rad from a slant that a classmate gave us. After gram staining them to make sure the samples looked good, we went ahead and got started. From E.coli pRAD1, we needed to extract the pRAD 1 plasmid and produce PCR product. To do this, we used the Zyppy Plasmid Miniprep Kit to extract the plasmid and then went on to do LongAmp PCR.  From Deinococcus radiodurans, we needed to perform a Genomic Extraction and produce PCR product. There was trouble finding the E.coli pRham so we did not move forward with it. From that bacteria, we knew we needed to perform a plasmid extraction and produce PCR product. Along the way, we made freezebacs of our E. coli pRAD1 and D. rad samples along with competent cells of our D. rad. 

Coming back from winter break, we were able to quickly pick back up from where we had left off. We were given E. coli pRHAM in the form of a freezebac for us to use to start our own culture. With this culture, we were able to begin plasmid extraction and PCR.

Methods: 

In order to begin a culture with this different strain of E. coli, we made 200 mL of LB broth and agar. It was autoclaved in order to sterilize it. The day we poured the plates, we heated up the flask on a hot plate and allowed it to cool until it passed the baby bottle test. This strain of E. coli requires us to add kanamycin to the broth in order to grow it because we are going after its kanamycin resistant plasmid. When the broth and agar mixture cooled down enough, we added 800 μL of kanamycin to the flask. We then poured the plates and let them set.   

When inoculating from freezebac, we needed to work under the hood. We put the freezebac sample on ice to keep it cold while we worked. The UV light was turned before we went into the hoo and we wiped all of our materials down in ethanol. We did a four way streak onto our LB + kanamycin plates. We did four plates in order for us to make sure we have enough growth and allow us to have duplicates. The plates were then put into the 37°C incubator for a day. 

After 24 hours of being in the incubator, the plates had grown enough for us to take them out and do a gram stain of each of them. The gram stains were consistent with what E. coli typically looks like. We were able to see gram negative (pink) rods. Since we were able to confirm that our plates were clear of contamination, we went ahead and inoculated from plate to broth. A few days prior, we had prepared several 125 mL flasks with 25 mL of LB broth and had them autoclaved. We added 100 μL of kanamycin to two flasks and placed them in the shaking incubator for a day.

On friday, we were able to do a plasmid extraction since we had our liquid culture ready. We used the Zyppy Plasmid Miniprep Kit. We followed the protocol as stated but before we began, we compacted the cells once. To compact the cells, we added 1000 μL of our liquid culture, spun it in the centrifuge for 1 minute and discarded the supernatant. When we finished our extraction, we checked our results on the nanodrop.  

Results:


ng/μL

A260/A280

A260/A230

KM 1 (Flask A)

29.6

1.90

1.00

KM 2 (Flask A)

24.7

1.98

1.30

MC 1 (Flask B)

6.9

1.54

0.31

MC 2 (Flask B)

7.2

1.53

0.64

Nanodrop results of E. coli pRHAM plasmid extraction 

Conclusion:

Two of our plasmid extractions (KM1 and KM2) were successful and had good numbers that we were looking for. The other two (MC1 and MC2)  had extremely low values and we decided that we will not be able to use them. We are planning on running the plasmids on a gel to confirm that we have extracted them. Once we have done that, we will be doing PCR with these plasmid samples. 




References 

ZyppyTM Plasmid Miniprep Kit. (n.d.). https://files.zymoresearch.com/protocols/_d4019_d4020_d4036_d4037_zyppy_plasmid_miniprep_kit.pdf

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