Team:St Andrews/Notebook

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Journal

Our iGEM Story


December-March

December 2011

Team St Andrews forms, uniting nine students, seven world class researchers and four PhD advisors from disciplines as diverse as Computer Science and Physics, to Biology, Medicine and Chemistry.

January - March 2012

Applications for sponsorship are made to specifically chosen businesses and organisations with an interest in advancing the Life Sciences. Very quickly, "BioSilta", "GenScript", "Clontech", "Geneious", "Integrated DNA Technologies" and "Thermo Fisher" pledge their support and Team St Andrews grows...

10 March 2012

National Science and Engineering Week explodes in Fife with a regional "Science Discovery Day". Team St Andrews works to convey fundamental concepts in Genetic Engineering and Synthetic Biology to members of the public in new and exciting ways. The interactive "Codon Game", the 3 Dimensional visualisations of DNA and DNA polymerase 3 and display "E. Coli: under the Microscope" are all well received. Children and adults alike are fascinated when DNA is extracted from bananas, using everyday kitchen utensils, before their eyes.

April 2012

Brainstorming sessions are held as the team researches project ideas. Some promising titles include:

  • "E. Coli and Omega 3: A Project to Feed the Minds of Our Generation"

  • "Enzymatic Methane Conversion in Cows: a Sweet Smelling Approach to Reducing Climate Change"

  • "Resurfacing Science, Resurfacing our Roads: Cell Factories and Metal Binding Proteins Recover Pavement Platinum"

  • "Project Bio-logic-al: Optimizing Soil Composition by Method of Biological Computation"

27 April 2012

Team member Josi presents "Spider Mutants and Bioterrorism - an Overview of Synthetic Biology as an Emerging Scientific Discipline" to a “TEDx” audience of over eighty scientists and non - scientists akin. Josi views the field as "ground breaking" and by the end of her talk, members of her audience too admit surprise at the wealth of possibilities that this new research area makes available. There is excitement at the tantalizing proximity of reality of these ideas.

May 2012

Project ideas are discussed in greater detail and are filtered until only two research topics remain. Those preferred ideas are: the production of Omega 3 Fatty Acids by E. Coli Cells ("E. Coli and Omega 3: A Project to Feed the Minds of Our Generation") and the production of Metal – Binding Proteins ("Resurfacing Science, Resurfacing our Roads: Cell Factories and Metal Binding Proteins Recover Pavement Platinum").


June

Week 1

4 June 2012

Full time work on Team St Andrews' iGEM Project finally begins! After an initial Group Meeting, two thirds of our student members continue in depth research into those project ideas generated previously; the rest begin work on Team St Andrews' Wiki.

5 June 2012

"CLC bio" and "Epoch Life Science" are the latest businesses to offer support to Team St Andrews. The former promises CLC bio Main Workbenches to members of the team while the latter offers products and expertise in DNA/ RNA preparation for molecular manipulation.

Team "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!

Team "Metal Binding Protein" weekly summary

"After a short meeting (which was dominated by the excitement of receiving our very own iGEM pins) we decided to split our iGEM family of nine into three groups. Josi, Veronica and Yiwang were to look into Omega-3 production; Constantine, Michael and I were to investigate Metal Binding Proteins and Antti, Alexey and Zoe were to focus on setting up the wiki.

The wiki team duly produced a sophisticated template.

The research teams, however, were unanimously agreed that biology is hard. Our motif quickly became standardised. Read, Think, Re-read, Re-think, Repeat.

After three days, our previously nomadic team settled in the University’s new Biomedical Sciences Research Complex. This proved to be a good move as it was followed by breakthroughs on all fronts. Metal binding peptides were located in their tens and the decision was made to try and express them on the surface of cells in display proteins as well as cytosolically. A membrane protein was found, preliminary peptides selected, and a primer design tutorial set up for the following Monday. All this success left us with one very important and so far unanswered question- how exactly are we going to assay all this?" (Team Member Hannah Taylor, Week One Report, 12/06/2012)

Week 2

12 June 2012: Team "Metal binding protein"

· Using E. Coli DH5-alpha: good at replicating vectors but not good at expression · Using pGEX-6P-1 vector with internal GST tag, selected with ampicillin http://ecoliwiki.net/colipedia/index.php/pGEX-6P-1 (further information via the “source” link) · Purpose: o First day: incorporate desired vectors into vector-replicating E.Coli and incubate them on selective plates to select out recombinant colonies with antibiotics o Second day: incubate the selected colonies in large quantities for vector harvesting o Third day: harvest vector from cell pellet thus pure vector miniprep.

"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.

Week 3

Omega Squad Week 3 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!

Week 4

"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.

Week 5

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University of St Andrews, 2012.

Contact us: igem2012@st-andrews.ac.uk, Twitter, Facebook

This iGEM team has been funded by the MSD Scottish Life Sciences Fund. The opinions expressed by this iGEM team are those of the team members and do not necessarily represent those of Merck Sharp & Dohme Limited, nor its Affiliates.