Parts

Toeholds

For our project, four biobricks were submitted to the registry, all of which are added as basic parts. Although two of the parts contain multiple parts from the iGEM registry, these have not been entered as composite parts. The main reason for this is that the inclusion of biobrick assembly scars in certain parts of the toehold structure could mess with the structure and function of the toehold, and would impose additional constraints upon the toehold design. Given this, we believe that it would be most useful to the synthetic biology community if this was characterised as a basic part which can be swapped between plasmid vectors as required, and the target changed using Q5 site-directed mutagenesis.

BBa_K1586000: Synthetic toehold riboswitch J23100 (GreenFET1J)

The first part submitted is a synthetic toehold switch which has been placed under the control of a standard constitutive promoter (J23100) and modified into a biobrick, namely through the removal of a PstI site at position 844. Figure 1 shows annotated features for K1586000.

Part K1586000 has been characterised and shown to work as expected, both experimentally and in silico. To see evidence of this, please visit the experiments page.

BBa_K1586001: Synthetic toehold riboswitch T7 (GreenFET1T7)

Also submitted to the registry is the GreenFET1 toehold structure, but under the control of T7. Like BBa_K1586000, it was modified into a biobrick, which included the removal of a PstI site at position 826. Figure 2 shows annotated features for this part.

BBa_K1586002: EsxB toehold riboswitch J23100 STOP (ZeusJSTOP)

Part BBa_K1586003 was a toehold designed with the aim of being used in diagnosis, but as with all toehold switches designed it can be applied in many different scenarios. As our project was concerned with showing the potential of toehold switches in aiding M.bovis diagnosis, the switch is designed to detect and become activated by EsxB mRNA. EsxB is a gene found in M.bovis, but not the BCG vaccine, and encodes for Esat6, which is involved in its pathogenicity. This toehold has been tested in silico, and will be further characterised at the University of Exeter after the competition has ended. To see data for this toehold, visit either the registry page, or the results page. Figure 3 shows annotated features for this part.

BBa_K1586003: EsxB toehold riboswitch J23100 (ZeusJ)

Part BBa_K1586003 was a toehold designed with the aim of being used in diagnosis, but as with all toehold switches designed it can be applied in many different scenarios. As our project was concerned with showing the potential of toehold switches in aiding M.bovis diagnosis, the switch is designed to detect and become activated by EsxB mRNA. EsxB is a gene found in M.bovis, but not the BCG vaccine, and encodes for Esat6, which is involved in its pathogenicity. This toehold has been tested in silico, and will be further characterised at the University of Exeter after the competition has ended. To see data for this toehold, visit either the registry page, or the results page. Figure 3 shows annotated features for this part.

Reporter characterisation: chromoproteins

Ideally, our diagnostic test would be carried out in the field with little technical equipment/training, therefore visualisation of the result must be simple. In order to do this, we decided that a colour change would be best, and that the expression of chromoproteins on activation of the toehold would be a good way to achieve this. In order to better inform ourselves and the model, and also to generate data which would be useful for testing toeholds with chromoproteins as the protein coding regions, we decided to further characterise some of the chromoproteins in the iGEM registry; amajLime, aeBlue, and eforRed. These parts were characterised by; recording each protein's individual absorbance spectrum to find their peak maxima, constructing a standard curve of protein concentration vs. OD at peak maxima for each chromoprotein, and determination of each chromoprotein' visual limit (i.e. the concentration at which the colour change is obvious). To see the rationale and results of this characterisation, visit the experiment's page.