During our work as an iGEM team this year, we received a complaint about a part that was designed by the 2017 iGEM Bulgaria team. This is part BBa_K2515002 (a gRNA expression vector), and our colleagues from the University said that, during their work with said vector, it has proven to work, but the cloning process itself is with very low efficiency. To check that, we decided to analyze the cloning of new gRNAs into this vector in a quantifiable way as a part of this year’s competition criteria fulfillment. To do that, we obtained oligos for three different gRNAs from ex-iGEM members from 2017 and used the following scheme:

We prepared DH10B competent cells and tested their efficiency using 300 pg of pSB1C3 (from the iGEM distribution kit). We made three independent measurements and estimated the transformation efficiency to be 4,75 x 106.
We performed 3 separate ligations with each gRNA into BBa_K2515002 (for oligo annealing and ligation protocol see iGEM Bulgaria’s 2017 wiki - section Protocols) and transformed equal amounts into our competent cells. BBa_K2515002 was digested with Eco31I and then split to the 9 ligations to ensure that the vector’s amount was identical in all reactions.
We obtained a large number of colonies for every ligation and then quantified the success ratio of the cloning procedure via colony PCR. For this PCR, we used one of the oligos from the gRNA and another one that anneals to the vector backbone. To obtain quantifiable data. We analyzed 2 separate petri dishes (20 colonies per each) per transformation and the final estimation was that the success ratio was 4,5/40 (gRNA 1); 5,8/40 (gRNA 2) and 3,2/40 (gRNA 3).
This quantitative data confirmed that the cloning efficiency of the used procedure is not high and one needs to check 10 colonies on average in order to find a single successful transformant. This data, plus details for the quantification process, were added to the part’s page in the Registry.
Having utilized a similar Eco31I cloning strategy for one of the parts from our project, we tried to optimize the cloning process by adding a red color-producing cassette between the two Eco31I sites. This cassette was created as a separate BioBrick with the number BBa_ K3208010. Amplifying it with the corresponding primers allowed us to insert it into our BBa_K3208000 part, creating an improved part - BBa_ K3208011. Next, we insert our cassette into the gRNA expression vector that we previously analyzed (BBa_K2515002). In this matter, we created an improved version of the vector - part BBa_ K3208012. We performed 3 cloning experiments (1 per each gRNA) and we found that when using the right color as a selection tool, we increased the cloning efficiency to the range of 4-6 positive colonies per 10 white colonies.
All quantitative data and part improvements were documented on the corresponding pages in the Registry.

Team:Bulgaria/Contribution

Project Contribution & Improvement