Continuation of Overlap Formation of a Linear Plasmid for Deinococcus radiodurans

Introduction:

Previous weeks have resulted in confusion surrounding multiple aspects of the overlap method for a three piece linear plasmid construction. Two protocols have been utilized thus far, Hilgarth being the primary focus for the most recent weeks given their undisputed success of overlap and minimalization of ghost banding due to techniques to increase specific binding and primer specificity for overlap and amplification steps. These techniques include using diluted primers, adjusting the template DNA loaded into PCR reaction, and utilizing touchdown PCR. That being sad, at the beginning of the semester, fragment isolations from D. radiodurans template DNA for the left and right, and isolation of tetr/a gene from an E. coli plasmid using standard PCR had been successful with minimal ghost banding, and overlap of left and tetr/a had been accomplished, thought not efficiently, with the Anna protocol. This week an attempt to use gel excised fragments of left and tetr/a assembled and the right fragment for overlap of the three part product was done, following Hilgarth protocol. After these results, we determined starting over completely from the beginning of each protocol was necessary to truly evaluate the products of each and how we should move forward with a greater chance of success. 

Methods:

Fragments that were isolated from gel and cleaned with both Monarch and Thermofisher gel excision kits were put into an overlap reaction in an attempt to observe some success even with insufficient loading DNA. This overlap reaction followed Hilgarth step 2 protocol for overlap and amplification was done with Hilgarth touchdown procedure. A 1.2% gel was run with 1x TAE with 5 ul of sample loaded per well. 

Plasmid extraction was accomplished after E.coli was grown for 72 hours at 28 degrees Celsius with no agitation in 10 ml of LB broth with tetracycline antibiotic. Standard Monarch plasmid extraction kit was utilized and elution done in 50 ul, and tested on a Nanodrop One. 

Both Hilgarth and Anna protocols were implemented to create new fragment isolations from template DNA, following step one from each protocol exactly. Anna was done at 60 degrees Celsius annealing for 30 cycles. Hilgarth protocol was only adjusted in touchdown temperatures, the 67 degree to 65 degrees Celsius and 59 to 57 degrees Celsius (no touchdown) to account for our fragment specific ideal amplification temperatures. 

All six fragments were run on a 1.2% gel and each well loaded with 5 ul of sample to verify band sizes and minimalization of ghost banding. From these results, the three fragments from Anna isolation protocol were loaded onto a 1.2% gel, with wells that were doubled in size by taping two wells together on the comb during gel setting. Each fragment had 45 ul of sample loaded, with the exception of the right fragment which only had 30 ul loaded. Each resulting band was gel excised and cleaned using the Monarch gel excision cleanup kit, and results were tested on the Nanodrop One. The left and tetr/a fragments post gel excision were input into eight different overlap reactions, identical conditions following the Anna protocol but with an additional temperature gradient to evaluate the possibility of higher overlap efficiency. The optimal is supposed to be 36 degrees Celsius, which had worked to an extent previously, so the subsequent gradient was ran from 34-44 degrees Celsius. 

Results: 

The initial gel from the overlap of left and tetr/a assembled and right from gel excision showed no results on the gel (Figure 1)

Plasmid extraction was successful with 48 ul of elution left with 303 ng/ul according to the Nanodrop One. 

The fragment isolation gel from both the Anna and Hilgarth protocols showed the three fragments on the left, (Anna left, Anna tet, and Anna right) were very distinct although not showing a very high concentration in the left and right, but all three had minimal to zero ghost banding. The Hilgarth fragments, the right three fragments (Hilgarth left, Hilgarth tet, and Hilgarth right) were riddled with obvious ghost banding, the least offensive being the left, but the right fragment showed signs of previous issues that had troubled us in prior reactions last week. It was yet again stuck in the well with significant smearing, indicating secondary structure of some kind. Tet was also heavily smeared and not as distinct in comparison to the Anna tet fragment isolated (Figure 2). 

Figure 3 shows the three Annna fragments loaded in high volume to prepare for gel excision. No bands besides the desired fragments are seen, but primer pooling is suspected (left, tet, right)

Figure 4 is the results of gel excision cleanup of the three Anna fragments to go into overlap PCR. 

                Ng/ul        260/280        260/230

Left            9.1            1.95            0.35

Tet            11.5            1.88            0.77

Right        5.6               1.93            0.03

Discussion: 

It has clearly been determined due to the results of this week that Hilgarth is not a viable protocol for our three part fragment overlap. It seems as though that touchdown PCR conditions are likely preventing us from getting decent results from that protocol, but it is difficult to say that the amount of template DNA loaded did not affect the fragment isolations as well. It seems that the Hilgarth team was likely working with fragments that had an overall higher working temperature, as opposed to our fragments that will amplify at 60 degrees Celsius but have homologous regions that prefer overlap at around 36 degrees Celsius, unless otherwise determined with the heat gradient completed this week. This determination was crucial for further success, and the Anna protocol will be used going forward entirely. Since we have previously seen the overlap of left and tetr/a from Anna in weeks past, it will be interesting to evaluate if a heat gradient can show a way to optimize overlap efficiency and produce a two part assembled fragment that can be added to the right fragment to complete the three part plasmid. Although we have had many difficulties, it seems as though success is on the horizon given that we know understand which protocol needs to be utilized for results. The greatest challenge may be the gel excision process that does decrease overall yield of fragments with only a 50% recovery rate, but there is a chance this step can be removed due to such a high efficiency of our primers with almost no nonspecific binding during fragment isolation.  

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.