Design of Plasmids

The first fundamental decision the team had to determine once selecting upon a project was the number of plasmids that must be constructed in order for the system to be operable. We had to be able to experimentally prove that the proteins were transacting, so had to take this requirement into consideration during the design phase – the sgRNA and dCas9 had to be located on different plasmids as they need to interact, whilst the fusion proteins must be located on a third plasmid as they must be able to interact with the sgRNA/dCas9 complex formed from the other two plasmids. This led to the decision to design three plasmids.

Another factor taken into consideration was the type of backbone that would be used for each plasmid. We identified the following aspects of synthetic biology that had to be acknowledged within the design:
- Each plasmid must have a different replication origin. If two had the same, competition between the plasmids would result in only one of the plasmids existing within a cell.
- Each plasmid must be resistant to a different antibiotic in order for individual plasmids to be selected for separately.
- The plasmid copy number must be suitable for it’s function.

Having reviewed these points, we selected pSB1C3, pSB3T5 and pSB4A5 as our backbones, with the plasmids having resistance to chloramphenicol, tetracycline and ampicillin respectively. It became apparent later in the project that there were possible issues with utilizing tetracycline resistance (see Lab Safety page for further detail) and the combination of pSB3T5 and pSB4A5, so we aim to alter our prototype model accordingly in the future. We’ve incorporated the sgRNA sequence into pSB1C3 because the high copy number nature of this backbone ensured high expression rates of the sgRNA, improving binding and therefore detection of our target substance. The fusion proteins have been designed as part of the pSB3T5 backbone, due to its relatively low copy number. This reduces the background expression of the reporter gene so that the change in expression (indicating a positive result) is more obvious. Finally, we used the pSB4A5 plasmid to carry the dCas9 and GFP parts. There is some conflict as to how this backbone will affect expression – according to previously published scientific literature, 4A5 is a low copy number backbone. However, when characterised with RFP and mini-prepped, it appeared to have high copy characteristics. Consequently, the dCas9/GFP plasmid could have a higher than desired background expression, but a greater increase in expression rate. On the other hand, if pSB4A5 behaves as a low copy number plasmid, the colour change would be less obvious and our detection system would be less effective. We had no issues when we utilized pSB4A5 for this purpose, with the apparent high copy number nature resulting in successful expression of the Cas9/GFP plasmid.

Experimental Proof of Successful Plasmid Construction

To confirm that our plasmids had the desired structure, the wet lab team performed a triple transformation of RFP in all three of them, plating them on the three corresponding antibiotics. Red colonies appeared on all plates, indicating successful existence of all plasmids together within the same cells.

Construction of Plasmids 1 and 2

We amplified target genes encoded on double stranded DNA ordered from, using pre-designed primers corresponding to the target gene. These primers were adapted to include Gibson or Golden Gate adaptors, so that an assembly reaction could be used to sub-clone the gene into a new backbone.

All plasmids carrying the inserted gene combinations were transformed into competent Top10 cells, except those with the sigma 54 fusion proteins. The Top10 cells used were appropriate for this task, as they had been made electro-competent and had a rapid growth rate. Sigma 54 fusion protein plasmids were transformed into Rpoz- cells, which, unlike Top10 cells, don’t produce the sigma 54 protein naturally. Our competent cells had poor transformation efficiency (around 3 x 105), hence when we discovered some issues with construction of the T7 variants, we invested in cells that had a transformation efficiency of 1010.

Transformed cells were plated, and multiple colonies of each variation were inoculated with LB. The colonies selected were those that did not express the red fluorescent protein, as this indicated that the target gene had been sub-cloned successfully. After extracting the plasmids from the cells by mini-prep , a purified sample and the relevant primer were sent for sequencing. We also completed diagnostic digests to gain a greater insight into how effective the construction had been.

Currently, we have managed to fully construct all of the following variants of Plasmid 2:
- Sigma 54 with MS2
- Omega with MS2
- Sigma 54 with PP7
- Omega with PP7
- Sigma 54 with COM
- Omega with COM

We are still working to successfully synthesize the T7 polymerase variants.

Construction of Plasmid 3

Creation of Plasmid 3 was hindered by numerous unsuccessful PCR reactions due to the formation of primer dimers in solution. The sgRNA cassette was amplified from the sgRNA g Block once the antisense primer had been redesigned using …., and Gibson assembly was attempted with a PSB1C3 backbone. Unfortunately, re-circulisation prevented the desired insertion. To resolve this issue, we utilized the compatibility of the biobrick prefix and suffix and the new plasmid backbone with the Xba1 and Pst1 digestion enzyme cut sites. Potentially, this ligation reaction may be affected by insufficient base length at the 3’ end of the sgRNA Pst1 binding site, which would increase the probability of the plasmid re-circularising. We combatted this by performing a digestion reaction using shrimp alkaline phosphatase, removing phosphates from the 3’ and 5’ end of the plasmid backbone. This prevents re-ligation of the two ends, encouraging ligation of the insert instead. Unfortunately, even when using this chemical and commercial cells, there was no significant increase in ligation efficiency.

Testing the System – Introducing the sRNA

In a digestion ligation reaction, we aim to subclone the sRNA (sensing RNA) gene, by using biobrick-compatible cut sites, into Plasmid 2. We selected Plasmid 2 (already encoding the fusion protein) as the host to illustrate that it is capable of transacting with the sgRNA plasmid.

Testing the System – In Vivo Formation of the RBP Handle

We plan to test three different variations of Plasmid 3 – 3 types of misfolded RBP handle in conjunction with the target site that resulted in the greatest GFP expression during the positive control investigation. To do this, we will transform all three versions of Plasmid 3 into Top10 cells. Having combined each of these with copies of Plasmids 1 and 2, we will monitor the rate at which the system expresses green fluorescence. Given that all the RBP handles are misfolded, GFP expression should be low (with exact values to be determined) as the binding site should not refold if the RNA being detected is not present.

Testing the System – In Vitro Formation of the RBP Handle

To complete an in vitro test, we will employ a version of Plasmid 2 that does not synthesize the sRNA, combined with each of the three versions of Plasmid 3 separately. Once this system has been freeze-dried onto a piece of filter paper, a concentrated solution of the RNA being sensed will be dropped onto the construct, and GFP expression will be measured. The sRNA (RNA being sensed) will be extracted from an external source, or ordered from a DNA synthesis company. Having gained a positive result during this experimentation period, we endeavor to accurately gauge the concentrations of sRNA required for complete saturation of GFP expression, and the minimum amount that still results in a significant indication. Determining these parameters will enable us to select the version of Plasmid 3 that causes the greater change in colour when sensing RNA is present.