Part:BBa_K2629003:Experience

Experiments were done on a plasmid in which the probe has been inserted, thanks the Gibson technic, in psB1C3-BBa_J04450.

Results of the clonning of the BFP gene instead of BBa_J04450 and the probe clonning

This is the alignments realized:

- between psB1C3-BBa_K2629003 and new BFP gene (A).
- between psB1C3-BBa_K2629003 and the probe after probe activation by PCR linearization (B).

Unfortunately, this is not the result that we expected. In fact, the sequencing shows that the cloning of the probe did not work. Regrettably, we did not have the time to make more sensibility tests. However, the substitution of RFP gene by BFP gene did work well as we can see one the figure A. This means that a new biobrick was constructed with the same RBS, promoter and terminator system as BBa_J04450 but with another gene than mRFP E1010 --> K592100 (BFP gene).

Test A: Is this part able to detect the target for which it has been designed ?

Unfortunately, results were too heterogenous to bring any conclusions. Indeed, the number of colonies, for the 1:100 and 1:200 ratios, expected was not good enought (there is a possibility that the detector was badly digested and was consequently transformed).

Test B: Is this part able to detect specifically the target for which it has been designed ?

In order to evaluate the specificity of the detector part of BBa_K2629003, different “false target” sequences have been synthesized. An algorithm was made by iGEM Grenoble Alpes 2017 to give random sequences, more or less homologous to the original target with 5%, 15%, 25% and 50% randomly modified pairs of nucleotides (length is kept 36bp).
The algorithm can be found here :

Unfortunately, we did not have the opportunity and the time to carry out these experiments.

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