Continuation of Three Way Fragment Assembly with Overlap, with a side focus on Deinococcus aquaticus Mystery 7 kb Plasmid

 Introduction: 

This week involved two major projects, one being the obvious continuation of three way assembly of three fragments, left, tetr/a, and right, for eventual transformation of Deinococcus radiodruans. As a brief reminder, this eventual overlap product will be used to hopefully knockout the LuxS gene within the chromosomal DNA. This proof of concept would be ideal for future experimental knockout in D. rad. So far, we had many issues with the right fragment correctly overlapping both with tetr/a itself and the combination fragment left and tetr/a. We would frequently see overlap product on gels that seemed to be stuck in the wells, possibly forming a secondary structure. This week we conducted a heat gradient of right and tetr/a, to isolate this combination and attempt three way assembly by adding the left fragment, which has been much more successful this entire semester. 

The other project pursued this week revolved around the mysterious 7 kb plasmid seen from Deinococcus aquaticus from sequencing data attained in the previous spring semester. Up until this point, it was highly debated as to whether or not this plasmid existed, especially with evidence that it may not have a clear origin of replication and no other known Deinococcus species has a plasmid anywhere near this size. If determined to be real, this plasmid would be a major tool that could be used in the lab to potentially transform other Deinococcus species much more easily. This week we attempted to isolate this 7 kb plasmid and perform restriction enzyme digestion to visualize it and begin to confirm its identity. 

Methods:

All gels were ran on 1.2% TAE agarose gels, with a 1 kb ladder and 5 ul of sample used for each lane unless otherwise stated. All PCR conditions unless otherwise stated included a total of 4:30 at 95 degrees, :30 of annealing, 1:00 to 1:30 of extension at 72 degrees, and 7:00 at 72 degrees Celsius of final stage. The MasterMix was kept constant with the exception of Q5 Polymerase, which varied between overlap step one and overlap step two, with step one having 0.5 ul per reaction and step two containing 0.25 ul per reaction. 

A heat gradient was run this week to confirm that right and tetr/a was not possible due at any temperature for PCR, the heat gradient from 35 to 60 degrees Celsius focusing on the overlap annealing temperature while the amplification of overlap product (step two) was kept constant at 60 degrees Celsius. 50 ng of right fragment and 35 ng of tetr/a was input, with 11 ul of MasterMix and the remaining volume made with PCR water up to 24 ul. The overlap was done with 15 cycles and amplification carried out with 30 cycles and the addition of primers 3 and 6. The results were run on a gel. 

After gel results, three temperatures determined most optimal were chosen and the remaining the 45 ul of each sample (35 degrees, 36.7 degrees, and 57.9 degrees) was run on a double welled gel for gel excision and cleanup. The resulting right and tetr/a products were used to attempt three way overlap by adding left fragment. These reactions had an input of approximately 50 ng of the 1700 bp right and tetr/a assembled product, and 40 ng of the 1000 bp left fragment. The four tested tubes were as follows, 54A- right and tet assembly at 35 degrees, three way at 54 degrees, 54B- right and tet assembly at 57.9 degrees, three way assembly at 54 degrees, 36A- right and tet assembly at 35 degrees, three way assembly at 36 degrees, and 36B- right and tet assembly at 57.9 degrees, three way assembly at 36 degrees. Each of the four were amplified in 50 ul reactions with primers 1 and 6 and ran on a gel to verify results. All samples were eventually run with the remaining 45 ul of volume in a double welled gel for an attempt at gel excision and cleanup. 

Two other samples were also ran to verify previous results and to confirm increasing overlap temperature would not be beneficial. Right and tetr/a was attempted again, one higher than the heat gradient at 63 degrees Celsius, and the other a replicate of 35 degrees which was seen in the heat gradient. They were overlapped and amplified identically to the heat gradient and again, run on a gel for visualization. 

Another heat gradient was conducted with the assumed optimal sample from three way assembly, which was tube 54A. Five samples rather than eight were ran, due to volume constraints, ranging from 54 to 62 degrees Celsius. This heat gradient focused on using the product from overlap reaction 1 of 54A and evaluating a heat gradient for the step 2 stage, amplification of that overlap product. Tube 1 was amplified at 62 degrees, 2 at 60.4, 3 at 58.9, 4 at 55.5, and 5 at 54. Tube 3 was unfortunately not identical to the other four, because the DNA input was only 2 ul instead of 4 ul like the other ones. The extension time was ran at 1:20 as a possible way to cut down on the presence of other undesirable bands seen on the previous gel. 

For the 7 kb plasmid, the first attempt included two samples from one D. aquaticus culture which was inoculated a week and a half prior by Alex and Kieran. Their project, which is attempting to lyse cells with lysozyme which was not previously accomplished by myself the previous semester, had been at least partially successful in their most recent trails. We decided to run one sample exactly according to the Zippy plasmid extraction kit, and one that included the standard lysozyme procedure as described by the paper Alex and Kieran based their experiments on. The Zippy kit called for 600 ul of culture, which was done for both procedures. The lysozyme protocol called for the isolation of cells by centrifugation, then two washes of 500 ul of Multibuffer which contains mostly detergents, and finally incubating the pelleted cells in 1 ml of lysozyme 1 mg/ul at 37 degrees Celsius for 5 minutes. This sample was integrated into the Zippy procedure at Step 3, which bypassed the Lysis solution and went straight to Neutralization solution. The resulting samples at the end of the kit, eluted in 30 ul, were run on a gel for visualization and tested on the Nanodrop for concentration. 

Another attempt was isolating the plasmid was made two days later, except two samples were ran both with the lysozyme procedures and a few modifications. Firstly, to increase plasmid concentration, 3 ml of culture was compounded to start with a greater mass of cells. Secondly, the lysozyme volume was adjusted to 600 ul rather than 1 ml, and the incubation at 37 degrees Celsius was increased to 15 minutes. Finally, the samples were directly introduced into the Zippy protocol, and did not skip the Lysis solution step. The two samples were done separately, with not cross contamination. These samples were again put on the Nanodrop for concentration, and then put on a gel. 

Finally, four more samples from the same culture were done identically to the most previous procedure described, with the exception that tubes A and B were combined in the column process and tubes C and D were also combined to further increase plasmid concentration. To help verify the identity of the bands previously seen, restriction enzyme digest was done on tube CD with enzymes XbaI and BamF, which are supposedly present according to the sequencing data. The resulting products should be seen as about 2400 bp and 4600 bp if it worked properly. Another control was done with tube AB, which included the enzyme buffer and no enzymes. These two tubes were incubated at 37 degrees for 30 minutes. On the gel, tubes AB and CD were run as controls (with no exposure to incubation, just directly from plasmid isolation) and AB with buffer as a secondary control, and CD with enzyme run as the experimental sample.

Results: 

 

Figure 1: From left to right: 1 kb ladder, 60, 57.9, 54.6, 50.1, 44.5, 39.6, 36.7, 35.0 (degrees Celsius). It can be seen there is overlap product of right and tet in each lane, the top prominent band in every lane. Samples at 57.9, 36.7, and 35 were isolated and gel extracted with the remaining 45 ul of samples and used for 3 way attempts. 

Figure 2: From left to right: 1 kb ladder, 63 degrees, 35 degrees Celsius. Secondary attempt at right and tet overlap, to confirm reproducibility. Does not look as clean as the heat gradient above, due to unknown changes, but overlap product still seen in each lane. 

Figure 3: From left to right: 1 kb ladder, tubes 36A, 36B, 54A, and 54B. 54B lane is the only one with bands and no significant smearing. The overlap product of 3 way assembly circled in red for clarity. 54A overlap 1 reaction was used based of these results for secondary heat gradient of overlap step 2 to try and cut down on the undesired bands seen in this sample. All of these samples were run with the remaining 45 ul and no bands were seen in any of the four samples, which raised some serious questions since gels were only run about 12 hours apart, so the three way product was no gel extracted. 

Figure 4: From left to right: 1 kb ladder, sample 1 and sample 2 from plasmid extraction both identically done with lysozyme for 15 minutes. Sample 1 has a single clear band at 7kb, as well as sample 2, which is much fainter but did have less concentration according to the Nanodrop. 

Figure 5: From left to right: 1 kb ladder, tube AB no incubation, tube CD no incubation, tube AB incubation with buffer, tube CD incubation with restriction enzymes. The first three lanes show two distinct bands, a mystery band above 10 kb, and a 7 kb band just like the previous gel. The last lane has the greater than 10 kb fragment, and then also has two other bands, one at approximately 4600 bp and a much fainter one at approximately 2400 bp.  

Discussion: 

Two major conclusion came from the work done this week, the first being that three way overlap is possible as seen in Figure 3, but does clearly need to be streamlined. It is very concerning that trying to isolate the three way product was unsuccessful, because it seemed overnight that tube 54A had bands on the gel and then didn't. Because there is a heat gradient that was ran on overlap 1 product of 54A, which has not been seen on a gel yet, it may be possible to accomplish gel excision next week but the reproducibility of the three way assembly is not certain at this point. The other major concern with overlap this week is the sudden success of right and tet assembly, considering nothing was changed in the methodology when doing the heat gradient in Figure 1 besides that the right fragment used was a freshly isolated sample which may have been slightly cleaner than the previous sample used, and that one person rather than multiple people did pipette work for the heat gradient. This leads us to believe that potentially human error in pipetting and maybe mixing up tubes had been leading to the inconsistent results we have been seeing, although I am not personally certain if that explains how right and tet have been consistently seen caught in the wells prior to this week. That being said, next week the results of the 54A heat gradient hopefully provide some clarity and can provide a gel extraction of the three way overlap product, and isolations can be done to go forward attempting transformation as soon as possible. 

The other major result was the visualization of what could be the 7 kb plasmid, nearly claimed imaginary earlier this week. The fact that we see in Figure 4 two different samples with a 7 kb band was very encouraging, but Figure 5 nearly confirms this plasmid in my eyes. Though faint, the restriction enzyme digestion seems to have worked, which leads us to ordering primers and attempting to replicate and sequence a region of this 7 kb fragment for certainty of its identity. This work will be done next week as soon as primers arrive, as the implications of this plasmid really existing is a major development for the lab. If it is real and functioning, then we can potentially open up a wide range of options for future projects that could involve transformation of aquaticus and other Deinococcus species. 

Sources:

Kikuchi, Masahiro, et al. “Plasmids in Several Strains of Deinococcus Radiodurans.” Radiation Research, vol. 139, no. 1, 1994, p. 123., https://doi.org/10.2307/3578742.