Colony PCR Amplification of 7kb Plasmid from Deinococcus aquaticus and cmr from E. coli's pRAD1 Plasmid

 Introduction 

This semester, Leilani Boren, Micheal Dockham and I are transforming the 7kb plasmid from Deinococcus aquaticus with E.coli's chloramphenicol resistance gene via PCR amplification and Gibson assembly. Once assembled, the 7kb plasmid will be farmed and reintroduced into D. aquaticus. To do this, we must first prime D.aquaticus' 7kb plasmid as our vector and E. coli's chloramphenicol resistance gene as our antibiotic resistance gene via PCR. 

DNA cloning works by inserting a target gene (cmr) into a plasmid (7kb) . Afterward, the plasmid is introduced into bacteria (E.coli) via transformation. Colonies will be screened via selective media (LB + chloramphenicol) and, once verified, will be used to farm recombinant plasmid DNA. 

Typically, Plasmid DNA is manipulated via restriction enzymes which will cut at cloning sites to create an area for our fragment to be introduced and hybridized. However, we will be using the Gibson Assembly due to successes in combining at least 2 DNA fragments without the use of restriction enzymes. 

Procedure

To begin, cultures of E.coli were grown in LB + 50ug/mL ampicillin overnight to ensure the pRAD1 plasmid was present; pRAD1 contains ampicillin resistance in addition to chloramphenicol resistance. Additionally, D. aquaticus culture was grown in TGY agar and broth. D. aquaticus will go through 2 25ul reactions of long amplification PCR while E. coli will go through 2 20ul reactions of short amplification PCR. 

To perform colony PCR on E. coli, 100ul of culture is incubated at 95°C for 10 minutes and cooled for 1 minute at 4°C before adding 4ul of boiled solution to prepped pcr tube containing 2ul forward primer, 2ul reverse primer, and 32 ul master mix. Once homogenized, 20ul of the solution was transferred into a separate PCR tube for a total of 2 20ul reactions.

Colony PCR on D. aquaticus is similar. 100ul of culture was incubated at 95°C for 15 minutes due to Deinococcus' extreme resistance to environmental stressors. This was then cooled at 4°C for 1 minute before adding 4ul of boiled solution to a pcr tube containing 2ul forward primer, 2ul reverse primer, and 42ul master mix. After homogenizing 50ul volume, 25ul was transferred into a separate PCR tube for a total of 2 25ul reactions. We used 59°C as our annealing temperature and 361 seconds extension time for a total of 30 cycles before the final extension at 65°C. 

Once PCR reactions finished running, samples were loaded on 30mL 1% agarose gels. 



Results






E. coli PCR Reactions E1 and E2 cmr band at ~660bp



D. aquaticus PCR Reactions A1 and A2 with Primer Bands


Discussion
To begin, the cmr resistance gene is around 660bp long. We can see bands around that area, but there seems to be a slight smear. Unknowingly, both gels were made with H2O and agarose rather than TAE and agarose, which may have produced the "banana bands" seen in the first figure. However, we are confident that we have obtained the cmr gene via colony PCR.

 No bands near 7kb were seen for the long amplification reactions. However, we can see primer bands in the lower region of the second figure. Three hypotheses are that the master mix may have expired, the annealing temperature is too high, and that the primers aren't working correctly. We predict that by using M23 primers as a positive control and lowering our annealing temperature to 55°C, we'll be able to conclude which variable to cancel out. Additionally, we will run 2 short amp reactions with boiled and isolated 7kb plasmid using M23 primers and 2 long amp reactions with boiled and isolated 7kb plasmid using primers for 7kb. 



Sources
Bergkessel, M., & Guthrie, C. (2013). Colony PCR. Methods in enzymology, 529, 299–309. https://doi.org/10.1016/B978-0-12-418687-3.00025-2
Nilsen, I. W., Bakke, I., Vader, A., Olsvik, O., & El-Gewely, M. R. (1996). Isolation of cmr, a novel Escherichia coli chloramphenicol resistance gene encoding a putative efflux pump. Journal of bacteriology, 178(11), 3188–3193. https://doi.org/10.1128/jb.178.11.3188-3193.1996






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