Continuation of Three Fragment Assembly for Deinococcus radiodurans transformation

 Introduction: 

The previous week revealed that the Hilgarth protocol could be disregarded going forward and the Anna protocol could and should be utilized for the greatest chance of success of the assembly of the three fragment plasmid. This plasmid, as a reminder of the eventual goal of this project, contains an antibiotic resistance gene that will hopefully be expressed after homologous recombination of this linear plasmid directly into the genome of competent D. radiodurans cells. Individual fragments were gel excised and cleaned in order to undergo overlap PCR again with left and tet, while the "optimal" temperature of overlap being 36 degrees Celsius suggested by Snapgene is simultaneously verified. Once left and tet overlap can be accomplished, then an attempt can be made for the full three part product needed for transformation. 

Methods: 

All gels were 1.2% agarose with 1x TAE solution unless otherwise stated, with a 1 kb gene ruler ladder. 

The gel excised fragments from the previous week were calculated to be equimolar, with 50 ng of total DNA loaded for the largest fragment. The left fragment was then 50 ng for overlap, and tetr/a was 65 ng. Eight tubes were made to a volume of 24 ul with 11 ul of MasterMix, the appropriate amount of DNA, and PCR water. They were ran on a heat gradient for 15 cycles from 34 to 44 degrees Celsius. Two microliters of these overlap step one reactions were used for amplification to visualize each product, with 10.75 ul of MasterMix and 1 ul of each primer (1 and 4) and PCR water up to 20 ul total volume. Amplification was done at 60 degrees Celsius annealing for 30 cycles. Five microliters of sample were loaded in each well for a gel, and evaluated. 

More individual fragments were isolated and gel excised identically to the procedure from the previous week in preparation for further trials. The Monarch gel excision kit was used as a repeat from last time as well to maintain consistency and hopefully further improve cleanup results. 

I was decided to attempt another heat gradient to replicate the one completed and extend the temperature range for further insight into optimal temperature of overlap for left and tetr/a. It was also realized the first heat gradient samples were not equimolar in fragment input, so the loaded masses were altered. The left fragment was still 50 ng total and the tet fragment changed to 35 ng. The gradient was also adjusted to a range of 34 to 54 degrees Celsius. Five microliters of these overlap reactions were put into amplification, again with 1 ul of each primer (1 and 4), 10.75 ul of MasterMix, and a total volume of 20 ul accomplished by additional PCR water. Another amplification of 60 degrees annealing and 30 cycles was done. 

Another single sample was attempted with all three fragments. 50 ng of both the left and right fragments and 35 ng of tetr/a loaded with 10.75 ul of Master Mix and a total volume of 28 ul. This overlap reaction was done at 44 degrees Celsius for 15 cycles. Five microliters of this reaction was added to 11 ul of Master Mix and 1 ul each of primers (1 and 6)  and amplification at 60 degrees for 30 cycles. 

Ten microliters of the second heat gradient trial and the single three fragment assembly attempt were loaded into each well for a gel to visualize all current products. 

Results:

Figure 1 shows the improvement in the gel excision cleanup process, as the concentrations of each fragment dramatically improved by three times for each fragment. These fragments were the input for the second heat gradient this week. 

Figure 2 is supplementary material for the evaluation of Figure 3, which the 1700 bp fragment can be faintly seen in sample lanes 1 and 2, as well as significant presence of undesired bands. Essentially all other lanes presented with little to no DNA to be evaluated. This was the first heat gradient of the week. 

The gel that that had the second heat gradient of the week and the single sample with all three fragments showed absolutely no presence of DNA or primers, so no gel is pictured. 

Fragment	Concentration (Ng/ul)	260/280	260/230
Left	32.0	1.99	0.51
Tet	33.4	1.92	0.51
Right	9.8	1.98	0.22

Figure 1: Second gel excision cleanup for each fragment 

Figure 2: Ladder key

Figure 3:From left to right: 1 kb ladder, 44, 43.2, 41.9, 40.3, 1 kb ladder,  37.9, 35.8, 34.6, 34.0 (degrees Celsius)

Discussion:

 There are many questions that came from the results of this week. The first question of the week coming from the first heat gradient which was done between 34 and 44 degrees Celsius, being that it was not understood why not very much DNA product was seen after amplification of the overlap products. Particularly in the lane which was done at 35.8 degrees Celsius because results had been seen in weeks prior at 36 degrees Celsius with left and tet overlap. In this gradient, 44 degrees was definitley optimal because overlap fragment was evident, which would indicate the need to evaluat even greater temperatures. This was the logic behind heat gradient number two of the week, which was essentially duplicate the first heat gradient and then also extend the temperatures higher to 54 degrees to be able to visualize if greater than 44 is really optimal. This leads us to the second great question of the week, which is why there was absolutely nothing to be seen on the second heat gradient (plus the "Hail Mary" sample). There was no evidence of DNA, in smears, in wells, or even in primers which can even be definitively seen in Figure 3. Initially it was thought that the wrong tubes, being overlap reaction 1, the overlap step with no amplification, were put on the gel but that was no the case. The MasterMix may have been bad, but that still does not explain the lack of primers on the gel. The entire amplification process may need to be repeated, even thought it is understood the initial overlap reaction may be the very problem itself. That being said, the strangeness of seeing nothing on the recent gel leads me to believe there was gross human error that led to these results. The process of removing the tubes from overlap PCR and transferring DNA to amplification immediately after was done very quickly due to time constraints and with many hands, so it may be possible that there was a mislabeling and mix--up of which tubes were which. At the moment, this is the only logical reasoning I can conceive the results, but next week will be aimed at redoing this heat gradient and the single sample attempting three way assembly, since these results are imperative in order to move forward. After this optimal temperature is found for left and tet overlap, another heat gradient very similar may be done to evaluate tet and right fragment overlap as well.