"ω-3" Weekly Summary

Research into polyunsaturated fatty acids has been active for over 15 years, with successes varying from creating transgenic plants enriched in ω-3 fatty acids to expressing an entire synthetic pathway from a gene clusters extracted from marine bacteria. We want to expand on this area of research by attempting to express a aerobic synthesis of unsaturated fatty acids in E.coli, which has never been done before. By combining the genes of the cyanobacteria Synechococcus and trypanosome T. brucei into E.coli, this vector should be able to synethise a unsaturated fatty acid up to at least 22-carbon length!

This week, our team has been focusing on preliminary research by reading relevant scientific papers and understanding the various pathways and methods of recombination. We’re focusing on groundwork research done in the early 1990s that appears to have fallen off the radar, and are excited to see where this path will take us!

"ω-3" Weekly Summary

This week, we took the first steps to put our grand scheme into laboratorial action! It also occurred to us that if we started calling ourselves DJ Omega 3 & the Fatty Acids, we would intimidate the other squad.

First of all, we chose a vector to work in, pET-15B. We started looking into promoters suitable for both the vector and the chain of genes we want to express, and figured out how to use the Registry of Standard Parts. The next step was to design primers for all four of the genes of our pathway - and that was certainly a steep learning curve!

Preliminary tasks done, let’s head to the lab! We carried out a transformation, growing the vector in a DH5-α strain of E. coli with ampicillin as the antibiotic marker. Well, we attempted to carry out a transformation. It failed, much to the glee of the Metal Mickies. Fortunately, we were able to use some pET-15b grown in E. coli for a different purpose. The colonies were allowed to grow, and we then carried out a mini-prep to isolate the vectors. Visualization was done by running the DNA on an agarose gel and using UV light. Once we were sure the transformation was successful, it was time to add the restriction enzymes!

Also, a PCR was run with the genomic DNA of Leishmania major, the trypanosomatids that are providing us with the 4th gene of our synthesis pathway. We ran 5 samples total: 3 using high-fidelity PCR at different temperatures, and 2 of Clontech’s PCR kits at 2 temperatures. We added the primers for Δ6 elongase (GenBank LmjF32.1160). This gene is around 1.200 kb large. The results show that the Clontech kit run at 48° C gave the strongest result.

"ω-3" Weekly Summary

This week has been a succession of PCR, digestion, PCR, PCR, digestion, vector growth, more PCR.

We can confidently say that we now know how to set up and run agarose gels! Looking on the bright side of things, we made a lot of mistakes these last few days that we hope to evade in the future: buffers need to be diluted, instructions followed, labels clearly written, and don’t lick the metal poles in the -80° freezer. Just don’t.

In order to learn all these valuable lessons, we had to sacrifice one thing: results. Many of our PCRs were unsatisfactory, plasmids did not digest properly, E. coli wouldn’t take up the vectors, and it turns out we have a knack for mysteriously making PCRs disappear on agarose gels.

Not until Friday after lunchtime did we finally have some successes: we found a PCR and the conditions that worked for the gene we are currently working on, the D6 elongase from L. major. Plus, we managed to grow a decent amount of vector and restrict it with enzymes. We hope that we’ve set a good foundation to build on next week – nowhere to go but up!

"ω-3" Weekly Summary

Squad Omega managed to create its very first preliminary BioBrick! We successfully inserted the L. Major D6 elongase into a pET-15a vector, and have sent it off to be sequenced over the weekend. At the moment, we are working on creating an assay to monitor this gene’s function. In order to do this, we have grown three different types of protein expression E. coli: BL 21 BL 21 codon stop, BL 21 plyS. After growing them, we will be analysing their natural 18-1 lipid content, in order to create a “background”. Once we have inserted our foreign proteins into our expression vectors, we will be able to compare the types of lipids synthesised.

We have also started working on another gene of our sequence, D15 desaturase from T. cruzi. Originally, we had planned to use this gene from the organisms Synechocystis; but as the genomic DNA is not due to arrive for several weeks, we have found a suitable, more accessible alternative. At the moment, we are running a PCR after designing primers to isolate the gene-of-choice.

"ω-3" Weekly Summary

We received our sequencing results from our preliminary BioBrick this week, and ---- nothing. The insert has a 99% protein match when using BLAST; unfortunately, this match is to a mevalonate kinase of a trypanosome that we haven’t been using. Some sleuthing in slabs and moving of freezers uncovers the sad truth: the vector we have been using since week 1 was not in fact empty. So 4.5 weeks of work amount to 4.5 weeks of gaining lab experience, but no more.

There is some good news among the bad, however. The genomic data we ordered for Synechocystis sp. won’t arrive until the end of July, but Dr Markus Gierth at the University of Cologne was kind enough to send us some of his colonies. Excited as we were to receive green smears, it also gave us additional DNA sources. After a Taq colony PCR, we ran high-fidelity PCRs to isolate our genes of choice. At the same time, we are also cloning D6 from L. major and T. cruzi. We hope that this simultaneous approach will give us fast results to make up for the time we have lost.

"ω-3" Weekly Summary

This week has been full of ups and downs for Squad Omega. We started off with a lot of hope when we found that the digestion of all necessary genes and plasmids worked. So we were ready to go for ligation and transformation into DH5-α cells. Much to our surprise, these transformations were then largely unsuccessful. However, we decided to pick some colonies from our plates and cultivate them to perform a miniprep. After analysis of our plasmids with numerous digestions and PCR reactions, and trying to re-do ligations, we finally were able to obtain some colonies containing our D15 gene, D6 and ELO6. D15 and D6 were sent off for sequencing, while for ELO6, we are still trying to confirm its presence in our colonies. Hopefully, next week we will be able to send off ELO6 and the missing D12 for sequencing, and start doing some protein expression.

Even though in the beggining of this week we felt like we needed a motivational speaker telling us not to give up, now we feel like we are finally getting somewhere and making progress.

"ω-3" Weekly Summary

"ω-3" Weekly Summary

This week we have been, once again, puzzled by science. After insertion of our Synechocystis delta-12 desaturase into our vector, we sent it off for sequencing. Result? Nothing. However, while waiting for sequencing results, we did some protein expression and analysed the lipid profile of BL21 cells containing our desaturase. Protein expression showed that the desaturase was overexpressed in BL21 cells, and these cells also showed a different lipid profile than background BL21 cells, with clear indication of desaturation. Thus, even though we have shown the protein is being expressed and having its own desaturase activity, we are not able to submit a BioBrick because, according to sequencing, we have nothing.

We have also started working with pET-duet vectors. We have ligated 3 of our 4 genes into specific cloning sites in this vector, hoping that we will be able to insert 2 genes simultaneously in the same vector in the future.