Continuation of Overlap of Linear Fragment for Deinococcus radiodurans Transformation

 Introduction: 

Last week provided insight to how we have been misunderstanding the Hilgarth protocol for overlap assembly of the three part fragment that will hopefully be transformed into D. radiodurans. It was realized that the correct amount of DNA may not have been loaded for the initial overlap reaction, which excludes primers. Both protocols, the Anna and the Hilgarth, stress equimolar inputs of fragments, as well as correct temperature conditions. Both of these factors have not been done correctly for our fragments, as the Hilgarth paper has fragments with homologous regions with an ideal temperature of 65 degrees Celsius, and our fragments are nearly half at 37 degrees Celsius. Therefore, following the Hilgarth protocol identically to the paper will very likely not yield results for our three part assembly. This week will focus on the Hilgarth protocol and establishing whether or not overlap of the left and tet assembled fragment with the right fragment is possible, and then purifying the fragment will be the secondary concern once assembly is visualized.

Methods: 

All gels mentioned were done at 1.2% agarose concentration with 5 ul of sample, 5 ul of PCR water, and 2 ul of dye used for each sample with a 1 kb ladder unless otherwise stated. All PCR reactions were done at 50 ul total volume with Q5 polymerase MasterMix. 

Step 1 Hilgarth protocol PCR condtions

Step 2 Hilgarth Protocol (overlap)

Step 2 Hilgarth protocol PCR conditions (overlap)

Step 3 Hilgarth Protocol (amplification)

Step 3 Hilgarth Protocol PCR conditions (amplification) 

One reaction tube was made which was supposed to follow Step 2 of the Hilgarth protocol, but followed Step 3 PCR conditions instead. The temperatures for touchdown in the PCR conditions were altered at Step 3 and Step 6, instead of 67 degrees to being 39 degrees Celsius and then 59 degrees being changed to 31 degrees Celsius to follow the same pattern. The amount of DNA loaded was based off of Nanodrop readings so the tube was again not equimolar since the left and tet assembled fragment still contained other bands. A gel was ran with these results post amplification with identical conditions to Step 3, no change in temperature. Two versions of this amplification was done, one with diluted primers (1 uM) and one with undiluted primers (10 uM) 1 and 6. This was to test the claims made by the Hilgarth paper that diluted primers highly increases primer specificity and produces cleaner results. 

Another gel was run after these results with the same products, but with heating each sample at 65 degrees Celsius and momentarily cooling on ice prior to loading onto the gel. 

Another gel was run from these results which was made with extended wells to accommodate larger loading sample, being 30 ul of the recently reamplified right fragment and the remaining 20 ul of the left and tet assembled fragment. Gel excision was done isolating the desired fragment from each lane, and cleanup with both the Monarch and ThermoFisher gel extraction kits was completed with an ending elution of 25 ul. One kit was used for each fragment to assess which kit would be preferred in future gel excisions. These results were tested on the Nanodrop 1. 

The two cleaned fragments were input DNA for Step 2 of Hilgarth in an attempt to overlap successfully with cleaner fragments. Not enough DNA was acquired post cleanup to follow the exact protocol of 100 ng/kb, but 54 ng of the right fragment was loaded and 42 ng of the left and tet assembled fragment was loaded to attempt equimolarity as best as we could. PCR conditions were altered for our homologous regions, with the first touchdown starting at 41.5 degrees and the second starting at 37 degrees Celsius. 

In anticipation of further work, two reactions were done using PCR conditions of Step 1 to amplify the left and tet assembled fragment, with undiluted primers 1 and 4, and results were ran on a gel to verify fragment cleanliness and identification. The PCR conditions were changed in touchdown temperature so instead of 67 degrees it was started at 64 degrees Celsius to better accommodate our primers with an ideal temperature at 60 degrees Celsius. 

Results: 

Figure 1 shows most of the DNA amplified in the reaction being caught in the well of the gel, with significant smearing also visualized below the well. This indicated either a very large unexpected band or a strange secondary structure being formed by the amplified product.  

Figure 2: Identical results to Figure 1, despite the heating method being used to discourage any unexpected secondary structures formed by the unknown amplified product. 

Figure 3: The left and tet assembled fragment which was amplified at 60 degrees Celsius with the Anna protocol (no touchdown and undiluted primers) did not reveal any surprising additional smearing or bands that was not seen on previous gels. The right fragment revealed much more smearing than was anticipated and the single band visible amidst the smearing was the desired fragment. 

Figure 4: The left and tet assembled fragment amplification with Hilgarth touchdown produced much more smearing and multiple undesired ghost bands that were not seen in the Anna amplification (also seen in Figure 3). The three band seen previously were also seen in this gel, but obviously not as cleanly.

Figure 5: The Nanodrop readings that went into the final overlap reaction, which both saw a significant loss of DNA and suboptimal cleanliness. 

Figure 1: Gel 1 from left and tet with right fragment overlap, with the wrong loading DNA and adjusted temperature conditions. From left to right: ladder, diluted primers, undiluted primers.

Figure 2: Identical samples to Figure 1 but with the addition of heating samples prior to loading onto the gel. From left to right: ladder, diluted primers, undiluted primers. 

Figure 3: Gel loaded with 20 ul of the left and tet assembled product (left) and 30 ul of the right fragment (right). The largest molecular weight band on the left and tet fragment was gel excised, and the primary band within the smear was gel excised for the right fragment.

Figure 4: The two products from Hilgarth Step 1 basic amplification of left and tet assembled, which had been previously done with the Anna protocol at 60 degrees Celsius. Both lanes are identical in conditions.  

Figure 5: Nanodrop readings of Gel excised fragments:

Fragment:                                        ng/ul             260/280            260/230

Left and Tet                                       5.6                  3.85                  0.01

Right                                                  2.7                  2.00                  0.02

Discussion: 

From these overall results in combination with the results from last week, the Hilgarth protocol may not be the ideal protocol for the assembly of our three piece fragment. Their two part assembly had overlap regions with a much higher temperature, and two of their three claims to success were not validated this week. The use of diluted primers results in very little product as seen in figures 1 and 2, which raises concern for future amplifications of overlap attempts. Not only this, but their touchdown technique was seen to produce much more smearing and ghost banding than the previously used Anna protocol as seen in Figure 4, to the point in which those samples are not even considered usable anymore. The Anna protocol is much more generalized and at very least has been confirmed to work with the limited success of the assembly of left and tet, and that products amplification. That being said, the results of the overlap reaction done with Hilgarth Step 2  with the gel excised fragments, though exceptionally low in DNA concentration and very dirty, has not been seen yet. Those results will decide whether or not we will essentially start from the beginning. It is suspected that if we did both the Anna and Hilgarth from the beginning now following the protocols correctly in every degree, then we would see success. The fact that we have made mistakes and did not understand the exact protocol at the beginning of using the Hilgarth may have led to its eventual poor results. Regardless, the touchdown and diluted primers will remain areas of concern, and there will likely by a higher reliance on the Anna protocol going forward. Figures 1 and 2 also raises questions as to how a very large fragment was amplified in those reactions with no indication of the desired 2700 bp fragment whatsoever. Since the heating method did not resolve anything in the secondary gel, it does not seem there are any possibilities of secondary structures forming preventing it from leaving the well. This is a mystery that will be considered in the restriction enzyme ligation team, if they also have similar results. If they do not see identical issues, it is a further indication that overlap has either an issue with PCR conditions or potentially leftover genomic DNA carried over from previous fragment isolations. If we do have to essentially start over with fragment isolations, we will be gel excising the original fragments from the beginning to confirm fragment identity in the later procedures and exclude the possibility of other undesired fragments or template DNA preventing success in overlap and subsequent amplification. 

Sources: 

1. Anna Behle 2019. Overlap extension PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.psndnde

2. Hilgarth RS, Lanigan TM. Optimization of overlap extension PCR for efficient transgene construction. MethodsX. 2019 Dec 4;7:100759. doi: 10.1016/j.mex.2019.12.001. PMID: 32021819; PMCID: PMC6992990. Modified GCC Biotechnology 2022.