Construction of a Linear Plasmid to Transform Deinococcus radiodurans

 Introduction:

 Deinococcus radiodurans  or D. rad is known for being an extremophilic bacterial species that can withstand high levels of oxidative stress and is incredibly efficient when repairing chromosomal DNA breakages. This bacterial species has been widely studied as of late due to these abilities, but it is the species of interest for this project for its potential uses in the future. D. rad chromosomal DNA contains genes for an AMC pathway to metabolize methionine. A part of the pathway, the LuxS gene, is critical for the pathway and is suspected to be integral for AHL production and maintenance. D. rad is also a species that no known linear plasmids currently exist in nature. The goal of this project is to knock out the LuxS gene of the AMC pathway by transforming D. rad with a linear plasmid consisting of three fragments (left, right, and tetr/a) which will replace the LuxS gene in the circular DNA and produce a bacterial cell that is terramycin resistant. This will prove the concept that linear plasmids can be transformed into D. rad  as well as recombined directly into the genome rather than being expressed from a plasmid. These transformed cells should be antibiotic resistant and appear sickly simultaneously due to the removal of LuxS. Each fragment will be isolated and bound by trial of multiple methods, so that transformation can be attempted. 

Methods:

Deinococcus radiodurans was inoculated in ten ml of  TGY media and grown overnight for genomic DNA isolation. The QUIAGEN UltraClean DNEasy kit was utilized with ten minutes of bead beating total in two minute intervals with one minute of ice incubation between each. Two washes with CB solution was also done to ensure cleanliness. Duplicate samples eluted in 50 μl and 100 μl and tested on Nanodrop for purity and concentration and stored at -20 degrees Celsius. 

Plasmid isolation was completed by Jonathan Hill from E. coli and stored at -20 degrees Celsius. 

PCR was run initially on all three fragments, left, Tetr/a, and right at 60 degrees Celsius annealing temperature. Total volume for every reaction was 20 μl  (16 μl of MasterMix, 2 μl of genomic DNA/Plasmid template, and 1 μl of each forward and reverse primer) unless otherwise stated. Several runs were also done with a 58 to 68 degrees Celsius gradient in which eight samples were prepared for each fragment. 

Agarose gels were ran for each PCR experiment with a one percent 1x TAE gel. A 1 kb ladder was also used each time, with 5 μl of PCR product loaded in each well. 

Results:

The gels revealed inconsistent banding in the tetr/a gene, but promising product concentration (Figure 1). The left and right fragments (Figure 2) were varied in their success. The left fragment was approximate desired bp length but needed further investigation for optimal annealing temperature. The right fragment was not successful. The products in Figure 3 of the tetr/a gene were increasingly distinct and in high concentration the more decreased the annealing temperature was. The left and right fragments with a heat gradient PCR (Figure 5) again produced regular but short right fragments. The left fragment still showed ghost banding but was also correct size and no significant effects on either fragment due to heat gradient. It was discovered that the right fragment and the tetr/a gene primers had been mislabeled, explaining the incorrect sizing of the right fragment. The tetr/a gene was repeated with the heat gradient with primers 5 and 6 which produced consistent products at the correct size as well as ghost bands below the desired band (Figure 4). 

Figure 1: The Tetr/a gene post PCR at with primers 3 and 4. Multiple bands present at undesired molecular sizes.  

Figure 2: (From left to right) Right fragment with primers 2 and 6, left fragment with primers 1 and 5, and 1 kb ladder. Prominent band from left fragment reaction at 1000 bp, with mild ghost banding. Right fragment is incorrect size and undefined. 

Figure 3: Tet r/a gene with primers 3 and 4 with a heat gradient (58 to 68 degrees Celsius) Lane 2 with 1 kb ladder, and left to right starting with Lane 4 Samples 68, 66, 64, 62, 62, and 58. The size of the band appears to be approximately 700 bp and the decrease in annealing temperature appears beneficial. 

Figure 4: Tet r/a gene with primers 5 and 6 with heat gradient during PCR. From left to right, 1 kb ladder, Samples 68, 66, 64, 62, 60, and 58. Very consistent and defined banding with regular ghost bands for every annealing temperature. Size appears to be about 700 bp. 

Figure 5: Both left and right fragments, left with primers 1 and 2, and right with primers 5 and 6. Heat gradient completed with each fragment. Lane 2 is 1 kb ladder, then left to right is heat gradient from 68 to 58 degrees Celsius left fragment, then heat gradient from 68 to 58 degrees Celsius right fragment. The left fragment is consistently the correct size, 1000 bp, with ghost bands present and increasingly worse at lower annealing temperatures. The right fragment is consistent with very minimal ghost bands but incorrect size. 

Discussion:

The gel results from the initial PCR and PCR gradient runs has solidified so far that the left fragment and the tetr/a gene are ready to be isolated and prepared for binding. It is undecided depending on upcoming runs whether products will be excised from the gels they were run on or if the product tubes will be directly utilized. Although there is significant and consistent undesired ghost banding for each of the two fragments, the desired products are significant enough to warrant success. The right fragment was no actually visualized with the correct primers for any of the reactions, which will be completed soon for progress to continue. The heat gradient had minimal to no effect for each reaction, besides the gel with tetr/a that was combined with the wrong primers. A wider heat gradient may be done in the future to observe tangible differences in how the annealing temperature affects these specific primers. The gels have likely shown human errors, the initial reactions on the right and left fragments did not have the correct primers for either fragment. Then in heat gradient reactions for the left and right fragments, the left fragment has two lanes that appear to have little to no DNA, indicating pipette errors prior to the reaction. Overall two of the three fragment have been identified and will be isolated soon, and the right fragment will be ran on PCR with the correct primers on a heat gradient to confirm hopefully similar results to the left and tetr/a fragments.

Sources:

Lin L;Dai S;Tian B;Li T;Yu J;Liu C;Wang L;Xu H;Zhao Y;Hua Y; (2016, February 25). DQSIR quorum sensing-mediated gene regulation of the extremophilic bacterium Deinococcus radiodurans in response to oxidative stress. Molecular microbiology. Retrieved September 9, 2022, from https://pubmed.ncbi.nlm.nih.gov/26789904/