Unfortunately, there are many issues regarding reagent logistics in Russia. We had ordered some competent cells from NEB in June, as a part of iGEM sponsorship, but received zero answers till the Giant Jamboree. We believe other teams may have similar issues, or have encountered them before. Thus we would like to share a protocol of competent cell preparation, using RbCl. There are already a number of such protocols on the Internet, however, the one we were taught at the Laboratory of Molecular Microbiology of the IChBFM SB RAS seems to be simpler and require fewer reagents. You can find the complete protocol on our Experiments page, here we will only provide evidence of its effectiveness.
Fig.1. Illustration of physiological aspect of LEAP2 and Ghrelin system .
Here you can see the result of successful heat-shock transformation with RbCl-prepared competent cells. Length is given in base pairs; each well is marked with a colony code. Bands of expected length are marked with its value "600", "500" etc. Note, that ligation reaction was performed using the blunt ends after the EcoRV digestion, and only the insertions with correct orientation were amplified. We repeated the transformation protocol twice with the same ligation and got decent amount of colonies with correct orientation insertion events each time. The transformation success was further verified using sequencing.
New information regarding LMWP
Despite the experiments of our team in particular not demonstrating a clear effect of LMWP on bacterial secretion, we still decided to mention this potential new application of LMWP in synthetic biology and add LMWP as a part of the registry. Perhaps it will inspire another iGEM team to build a project around LMWP, just like it has inspired us. You can find new information added at the LMWP part page in the registry: BBa_K380002
LEAP-2 part creation
We created a standard biological part coding the LEAP-2 protein and added some design notes if one would like to use or re-design it. K4086000
At some point in our iGEM journey, we organized the August School of Synthetic Biology which gathered students from all over Russia interested to provide them with new knowledge and advance their laboratory skills as well. So here we’d like to share with you some of our educational materials that can be helpful for such an event if you are ever planning to organize one.
link 1 – Protocols for plasmid DNA isolation, determination of DNA concentration, bacteria transformation.
link 2 Protocols for TA cloning, colony PCR, restriction product analysis.
link 3 – A presentation aimed at high school students about synthetic biology.
Our protocols include not only the particular steps of the procedures, but also have questions and dedicated free spaces for students to make notes and reflect on their results. You can adjust these files as you see fit, so that they would be suitable for your event.