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Experiments

Cloning

We designed our gblocks by using the Snapgene software and then ordered both these gblocks and primers from IDT. Some of our constructs proved difficult for IDT to make so we decided to break them down into samller pieces whcih would reconnect in the lab. When these arrived they were resuspended using the appropriate protocol from our protocols sheet.

Our initial method to insert our DNA into the pSB1c3 backbone was to digest our both the backbone and our DNA using the same restriction enzymes and then ligate the fragments together. As our source of pSB1c3 we chose to use a stock of the plasmid containing the insert NH75 left from the previous team. The reasoning for this was so that with the circular plasmid we could immediately transform and grow up to create a stock.
Before we could begin digesting our DNA we need to amplify the concentrations by PCR. For our 12 gblocks to begin with we had designed individual primers which extended out of the Biobrick prefixes and suffixes to try and ensure there would be no non-specific binding/contaminants would not be amplified. Here we also tried to reconstruct some of our gBlocks that IDT could not synthesize by carried out overlap extension PCRs. Unfortunately we struggling to find conditions which led to successful amplifications for some of the parts so we resorted to using the smaller standardized primers which only corresponded to the biobricks. After running some gradient PCRs scaling both temperatures and DMSO content we found that for the majority of our gblocks, 5% DMSO, 60 degrees were the optimal conditions.
After PCR, the correctly amplified fragments were separated by running on 1% agarose gels. We had difficulties in successfully extracting a high concentration of DNA from our gels but these were resolved once when we began using a different extraction kit.

Digest & Ligate

During the first attempts to digest and ligate our constructs we found upon transforming, that we had many false positives from linearized plasmid being reattached without our inserts. This led to us treating our vectors with antarctic phosphatase prior to ligating. This eliminated our false positives but due to unknown reasons we could not succeed in constructing the intended vectors. This led to us considering alternative methods of getting DNA into vectors

Gibson Assemblies

After failing with traditional restriction enzymes we decided to design new primers for our constructs that would allow us to attempt the assemble them via a Gibson Assembly. Gibson assemblies involve having complementary sections at the ends of DNA fragments which are then exposed by an endonuclease, allowing them to anneal together and be ligated. Despite initially showing promise this method also failed to prove fruitful for us, causing us to move on

Site-Directed-Mutagenesis

This versatile method allows you to insert/delete or mutate fragments of circular DNA and over the course of the project we made use of all 3 avenues. To rid our stocks of the initial insert NH75 we firstly carried out a deletion on the stock of pSB1c3, leaving no constructs between the biobricks. Later in the project when we began working on kill switches we designed primers that would insert uni and bidirectional promoters upstream of the endonucleases we were improving from Oxford’s 2016 iGEM team. A mistake in designed these primers resulted in us creating an invalid bidirectional promoter due to an EcoRI site being present. This was corrected by a single base mutation by SDM.
Another way we used SDM was to attempt to test a novel method of introducing large sections of DNA into vectors without having to use the above methods. The length of DNA that can be inserted by SDM is generally limited to around 100 bases due to the ability to make primers long enough as non-specific binding becomes a larger issue as the primer length increases. Currently, to consecutively carry out insertions in this manner would be slow due to the need to transform, culture and miniprep before another SDM could be attempted. Our method to bypass this would be to design primers in such a way that the first treatment would introduce a restriction site into the vector which would be removed by the second round of SDM. This would allow you to take the vector product of the first SDM and immediately SDM again. Treating this product with the restriction enzyme would linearize any remaining product from the initial SDM ensuring only the intended vector will be transformed, consequently doubling the rate at which insertions can be conducted.

Labelling System

It is important in the lab to know what you’re working with but also be efficient with your time. Hence we created a few cheat sheets to speed up preparing PCRs as well as labelling all our constructs. We printed these nifty tables out and hung them up in the lab!

Section 2

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Section 3

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