Team:GeorgiaTech/September

Submitted our track selection, project abstract (which completely changes later), our team roster, and our safety questions. For our track selection, we chose 1) New Application, 2) Health/Medicine, and 3) Foundational Advance.

We had a meeting with our advisers where considered our final experimental design and our final options for the biobrick. With the September 28th deadline, everyone was concerned about making the deadline to submit. Our options were to PCR cas 9, PCR CRISPR, or synthesize the repeat region. It was also discussed that we could not submit any DNA from S. thermophilus DGCC7710 due to a nondisclosure agreement for material transfer.

We placed an order for genomic DNA from LMD9 to amplify CRISPR out of it and hopefully clone it in a vector which could then be inserted into E. coli cells.

We designed primers to amplify out the entire 9kb locus of CRISPR out of the LMD9 genome and ordered them. They should arrive at the same time as the LMD9 genome. Also, as a team we decided to design primers to amplify just the Spacer/Repeat region which is around 1.4kb out of the LMD9 CRISPR genome. We noticed that there is an SpeI site present in the Spacer/Repeat region of the CRISPR and decided to find various ways to solve it.

We received our LMD9 genome from ATCC and our primers along with it. The primers have EcorI + XbaI site present going forward and SpeI + PstI sites present in the reverse primer. This was done to allow us to ligate our 1.4kb insert according iGEM biobrick standards. To solve the problem of our SpeI site at a distance of about 500bp from the leader sequence (the beginning of the Spacer/Repeat Region), we decided to use the method of Site-Directed Mutagenesis (SDM) as suggested by our advisers.

We PCR amplified out the LMD 9 Crispr Spacer/Repeat region out of the genomic DNA and ran it on gel. We obtained bands at 1.4 kb, as expected.

We started the process of cloning our insert in the TOPO cloning vector as the TOPO vectors are more easy to subject to SDM. For its specific protocol, refer to our protocols section.

After incubation period of about 16 hours, we picked 10 colonies from the plates for mini-prepping the vector which hopefully would have our insert. The mini-prep was performed on all the ten colonies and the concentration results observed are as follows:

We performed double digest on the 8 plasmids we obtained from our miniprep to verify whether the TOPO vector consist of our Spacer/Repeat Region insert. We used the EcoRI and PstI restriction sites to double digest. After obtaining the digest,we ran it on an agarose gel to verify whether the insert is present, but the results were inconclusive. We decided to continue with the SDM hoping that the insert is present.

Followed the complete protocol for SDM as described on our protocols page. Tranformed and plated the bacteria and waiting for their growth.

No growth observed with the SDM bacteria. We decided to revert to our backup plan of cutting our insert at the 500 bp region around the SpeI site and use that as our insert for the biobrick. Digestion and gel purification yielded 5.6 ng/ul of insert DNA.

The bacterial transformation failed. Transforming biobricks again in the Novablue cells and hoping for growth.

Observed colonies with pSB1C3 and innoculated them and performed miniprep on them. Digest the plasmid and do a gel purification. Ligate our insert in the plasmid vector and transform Novablue cells with it.

Check for growth.... Cultures were centrifuged in the hopes that we could perform a miniprep and get DNA in time to ship it, but minimal growth was observed at 7PM. We checked the cultures again at 10:30PM and observed a significant amount of growth, but unfortunately it is too late to proceed. Hopefully next year will be better! :’(