Click to download the SeaDNA protocol.

Click to download the SeaDNA Slime Antimicrobial Properties document.

Week 1: 02-June-2014 to 06-June-2014

As we came up with the idea for the SeaDNA project in March, we used this week to fully design our wetlab protocols and schedules. We also ordered the Hagfish cDNA from Gen9.

We also started working with Indie Bio, a synthetic biology accelerator, and began learning about synthetic biology start-ups.

Week 2: 09-June-2014 to 13-June-2014

We received lab training and learnt how to perform basic molecular biology skills and practicing protocols outlined that are included in our lab design. E.g. Gel electrophoresis, running restriction digests, SDS-PAGE and DNA extraction.

We began the completing the various types of paper work that is required to incorporate a company in Ireland.

Week 3: 16-June-2014 to 20-June-2014

We continued the lab training learnt how to analyse data and present that data. At this stage we were ready to start the lab work on the SeaDNA project.

All incorporation paperwork was completed and we began making a presence in the media. This included working on a company website, press releases and contacting the local newspapers.

Week 4: 23-June-2014 to 27-June-2014

We began the lab work by digesting the pRSF and pCDF plasmids using Eco RI and HindIII. These plasmids were then run on agarose gels. The first trial showed very little DNA on these gels, so the restriction digest was repeated for both plasmids. The second trial produced much better results, so the DNA was extracted from the gel.

On the 24th of June, Benthic Labs Ltd was incorporated, we also began attending guest talks from various world famous business gurus, and one of them was Thomas Gad. We also set up a company business account.

Week 5: 30-June-2014 to 4-July-2014

The pCDF and pRSF cut backbones were ligated with a random insert to see if any colonies grew when they were transformed. This was also repeated a second time.

We also began fine tuning the sales pitch and elevator pitches and practising for the half way demo day that was set up by Indie Bio, which would happen the following week.

Week 6: 7-July-2014 to 11-July-2014

This week, we spent working on business plans and practising sales pitches. On the 4th of July, Benthic Labs Ltd were lucky enough to give their sales pitch to potential investors, Bill Liao and Sean O’Sullivan from SOS Ventures.

Week 7: 14-July-2014 to 18-July-2014

The Hagfish DNA arrived, the DNA was digested and a small portion of the sample was run on an agarose gel to verify the insert size. It was then stored at -20 degrees Celsius. Primers were ordered for both the sequences.

Week 8: 21-July-2014 to 25-July-2014

A PCR was performed this week to amplify the DNA from the Hagfish. This gave us a reserve of DNA to work with in the future. We also ligated and transformed both the alpha and gamma subunit separately, using the pCDF backbone into competent DH5α E. coli cells. After incubating plates overnight, colonies were found on both plates.

Benthic Labs Ltd was focusing on increasing a media presence by staying active on all social media sites, we also contacted journalists from various local and national newspapers and radio shows.

Week 9: 28-July-2014 to 1-August-2014

The colonies were isolated and grown into a culture overnight, these cultures were then used to do plasmid preps to isolate the pCDF alpha subunit, and pCDF gamma subunit cloned plasmids. The isolated plasmid was then digested again with Eco RI and Hind III, an agarose gel was performed to verify that the correct subunits have been inserted into the backbone. The isolated cloned plasmids, were then separately transformed into BL21 E. coli cells givibg a successful transformation as there were colonies on both plates.

Week 10: 4-August-2014 to 8-August-2014

Colony PCR was performed on both cloned samples, this verified that the transformation was indeed a genuine result and not a spurious contamination. Plasmid preps were then performed on the successful colonies and the cloned plasmids were isolated.

Week 11: 11-August-2014 to 15-August-2014

We began the purification of the His-tagged protein which took place over 3 days, this included making both broths and cultures, inducing IPTG expression, purification of the His-tagged protein using column chromatography and analysing samples in an SDS-PAGE. By viewing our gels we clearly saw distinct bands that suggested the expression of both the alpha and gamma subunits. These proteins were insoluble when induced at 37 degrees.

On the 12th of August, Benthic Labs Ltd were featured on University Cork College’s website, see link here.

Week 12: 18-August-2014 to 22-August-2014

As the purification and expression of the protein the previous week showed that it was insoluble at 37 ̊C, we repeated the experiment at 30 ̊C. However the results remain the same.

Benthic Labs was featured in an article in the Irish Times which is a national newspaper, to read the article please see this link. On August 19th, Benthic Labs attended the first ever synthetic biology demo day in Dublin, Ireland where we gave our sales pitch in front of various investors.

Week 13: 25-August-2014 to 29-August-2014

The insoluble protein pellets were then analysed and compared to the natural hagfish slime which we had been working on simultaneously.

Benthic Labs was now focused on continuing this project, the first target was upscaling the product, this involved liaising with various lecturers in the university so find out if they would support and advise on the lab work behind the project.

Week 14: 1-September-2014 to 5-September-2014

We prepared the plasmids required for Biobrick assembly for iGEM by digesting the subunits with the enzyme SpeI and Eco RI, and digesting the given plasmid with the same enzymes. These digestions were then followed by a ligation reaction and transformation to bulk up the plasmid and screen for successful colonies.