Continuation of Three Way Assembly and Isolation of the 7kb Plasmid

 Introduction:

This week there were multiple attempts to make progress with the recently assembled three way fragment that had been the focus of this semester. In an effort to optimize the three way assembly process, we in fact had trouble actually replicating the results from the previous week and led to several PCR reactions as well as a heat gradient that were all unsuccessful. Seeing as the proposed three way assembled fragment could not be gel excised then we are again at a standstill for the total assembly process. The 7kb plasmid from Deinococcus aquaticus was isolated again in an effort to conduct a single digest experiment to validate the identity of the 7kb fragment of DNA yet again, and provide samples for Jonathan to amplify a roughly 1100 bp section of the plasmid. A transformed D. radiodurans was also put through the plasmid isolation protocol to isolate a 6800 bp plasmid, so that the ampicillin resistance gene it included could be amplified as well to validate its identity. 

Methods: 

The amplified reaction tube that showed successful three way assembly the previous week had a remaining 45 ul of sample, which was loaded onto a 1.2% gel with 1x TAE. The other three samples which had been shown as unsuccessful, only smears, were also run identically in order to try and find the three way assembly band to gel excise. Gel excision was not possible due to an overloading of the gel, and no bands were visible in any of the samples so three way assembly would have to be amplified again. 

The overlap reaction tube that had been amplified to show three way assembly on the gel was amplified identically to how it was done previously, with 4 ul of sample in a 50 ul reaction and 1 ul of each primer (forward and reverse, 1, and 6). This repeat sample was done amidst a small heat gradient testing 54, 55.5, 58.9, 60.4, and 62 degrees Celsius. This was run on a gel as well to attempt gel excision, but was again unsuccessful for every temperature. 

Another heat gradient was done between gel excised right and tet assembled fragments with the left fragment, from 30 to 64 degrees Celsius. 50 ng of the right and tet fragment were loaded, and 30 ng of the left fragment to try to be equimolar in the reaction. The samples were all amplified at 60 degrees Celsius and visualized on a gel. 

The 6800 bp plasmid from D. rad was isolated and ran on gel using an identical process of multibuffer, lysozyme with incubation, and the standard Zyppy kit, except for using another lysozyme solution. After these results, two more samples were done with not transformed cells in order to confirm plasmid isolation validity.  D aquaticus was also isolated but encountered issues with low DNA concentration likely due to another change in the lysozyme or an unknown change in protocol. 

Results: 

Figure 1: overlap heat gradient of right and tet assembled with left with a 1 kb ladder. Identical faint smearing across every temperature from 39 degrees to 64 degrees overlap temperature. All amplified at 60 degrees Celsius.  

Figure 2: From left to right: D. aquaticus A, D. aquaticus B, D. rad with no plasmid A, D. rad with no plasmid B, 1 kb ladder. One aquaticus sample showed a band but at greater than 10 kb, and some likely genomic DNA in the well. The D. rad samples showed faint smears but no plasmid bands at any size which validated previous results. 

Figure 3: D. rad plasmid extraction with transformed cells with a plasmid of 6800 bp. No bands at 6800 seen, only greater than 10 kb and then likely genomic DNA in the well. 

Discussion:

Unfortunately there was no success to recreate the three way assembly, which poses many questions as to how a sample can be successful and seemingly not overnight. The amplification of the exact same overlap tube that produced a band at the correct size resulting in  nothing but a smear is very concerning because these results are clearly not reproducible, especially considering all the other attempts this week to see any kind of success or any bands whatsoever. For all intents and purposes experimentally, no three way fragment was ever assembled because it happened only once and could not be done again. It may be more useful for resources to be put towards outsourcing the three way assembly so the project as a whole can move forward with an attempt at transformation. 

The 7 kb plasmid as well as the 6800 bp plasmid were able to be isolated but not as nicely as it was last week, but the positive information from Jonathan being able to successfully amplify both ampicillin and the 1100 bp segment out of the 7 kb tells us that the prominent bands slightly greater than 10 kb on the gels are likely just dimerized versions of each plasmid, The newest lysozyme is a potential cause for the recent poor plasmid isolations, but that will be looked into shortly so that a definite protocol is resolved for plasmid isolations, which we will undoubtedly be using in the future for new experiments.