Team:Bristol/Experiments
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Interlab Study
The first few weeks of our Wet Lab work was spent completing the Interlab Study. You can read more about this on our Interlab Study page.
PCR for Nap and Nrf fragments
The first stage of our Wet Lab work was to optimise the Nrf and Nap operons to be BioBrick compatible. This involved removing specific restriction sites, adding spacers, add most importantly adding the BioBrick prefix and suffix (more detail on our Design page). As these modifications would have taken significant time in the lab, we decided to get our constructs synthesised by IDT. After waiting a few weeks, we were informed that the synthesis was not possible. Therefore, we split each operon into 4 fragments (made to be easier to synthesise), which we could assemble ourselves using Gibson Assembly to obtain the full constructs.
On receiving our Nap and Nrf fragments, we first had to amplify the fragments by PCR to have enough template for Gibson assembly. Unfortunately this was not as straight-forward as it first seemed. It took us a total of 1 month to achieve clean, purified products of all of our fragments, apart from Nrf3 which still presents problems. Overall, three weeks of this stage was spent optimising the PCR for using Phusion polymerase (Fig. 1).
Fig. 1: Agarose gel of all amplified Nap and Nrf fragments and the pSB1K3 vector used for cloning. 1 kbp ladder (NEB).
Multiple Gibson Assemblies of Nap and Nrf fragments were attempted with the Phusion-derived PCR products, however these consistently resulted in colonies with only re-circularised plasmids (as determined by colony PCR). With only a few weeks remaining, we decided to move away from Gibson assembly, and attempt overlapping PCR instead to combine our fragments.
Overlapping PCR
We attempted overlapping PCR with all fragments in the reaction together, however this yielded no results. Therefore, we decided to use a simpler, sequential method, combining only two fragments at a time. We managed to assemble Nap1 -DT and Nap2 using this method (Fig. 2), however due to non-specificity of our primers, many other incorrect assemblies were present in the mix (Fig 2.A). Even after gel extracting our band of interest (Fig 2.B), and amplifying this with PCR, the nonspecific bands returned (Fig 2.C), preventing us from continuing with this method.
Fig. 2: A) Agarose gel of overlapping-PCR products. Combinations of fragments are indicated at the top of each lane. Differing annealing temperatures for duplicates of Nap12H and Nap12-DT are indicated. The band circled in blue is the expected size (~ 3.7 kbp) of assembled Nap1-DT (1.8 kbp) and Nap 2 (1.9 kbp). 1 kbp ladder (NEB). B) Agarose gel of the total remaining product of Nap12-DT overlapping PCR (60°) for gel extraction. Band to be excised is highlighted in blue. C) PCR of the product from gel extraction. Nap12-DT is highlighted in blue, and all other bands are nonspecific products.
Discovering Ultra
We then tried using different polymerase enzymes, including Ultra and HiFi. Ultra worked much better than Phusion with most of our fragments. Fig. 3 shows amplification of our fragments using Ultra, after purification.
Fig.3: Agarose gel of all amplified Nap and Nrf fragments used for cloning. Ultra polymerase was used for purification and fragments were subsequently purified (see Protocols). 1 kbp ladder (NEB).
