Possible Project Ideas
Diary: Week 1
During week 1 the team decided on a rough project outline which involves producing a protein microcompartment found in Salmonella in E.coli. Bacterial microcompartments (BMC’s) are protein organelles found within bacteria. The physiological function of these compartments is to promote specific metabolic processes by colocalising enzymes with subtrates and also cofactors. After expressing the microcompartment in E.coli we will attempt to target different proteins to the compartment which may have a bioremediation function for example. Ideas on what could be targeted to the compartment as well as a name for the project are still under discussion.
The team was given a health and safety briefing and before starting in the labs attended a several tutorials on basic microbiology and molecular biology. The rest of the week was spent learning different laboratory techniques such as miniprep, transformations, isolating chromosomal DNA, PCR and restriction digests which will be critical for our project. Although this was largely successful some of the team did not get the desired result from their PCR’s (Jane and Brian) better luck next time. Towards the end of the week we had a group meeting and discussion to establish a cloning strategy and also to design the primers needed for our gene of interest. Our computer team spent a large amount of the week designing our website which is now up and running.
Fail of the Week = Jane’s PCR
Diary: Week 2
Week 2 saw the team begin the iGEM project. It began by performing mini-preps to extract the plasmid pUNI-PROM from our E.coli strain. A restriction digest was then performed as well as adding an Antarctic phosphatase to prevent our plasmids rejoining. Our plasmids are now ready to be incorporated with our genes of interest. While waiting for the primers to arrive our team divided into groups to work on a project outline and description as well as the health and safety issues of the project.
Primers finally arrived. PCR’s were performed and our genes of interest were successfully incorporated into our vector through ligation and stratoclean steps. Our clones were left to grow in LB Amp medium so that we can perform Quick change on the genes which have additional Pst I sites on them.
Fail of the week = Natasha and her butter fingers
Diary: Week 3
This week the iGEM spent their time sequencing and verifying that that our cloning of individual pdu proteins as well as the quikchange was successful. As well as this the team began to work on the targeting sequence of the protein PduD. It is known that PduD contains a 40 amino acid signal sequence which is necessary to incorporate proteins into the microcomparment. As well as using a 40 amino acid sequence we are also going to attempt to use a 20 amino acid sequence. Towards the end of the week we began to ligate our different Pdu proteins together into a single pUNI-Prom plasmid plasmid beginning with PduAB-TU.
Fail of the Week = Rachelle’s ‘incompetent’ cells and Lucy’s transformation
Diary: Week 4
After PduAB-TU failed to ligate the first time or cloning strategy was adjusted so that we ligated AB and N together firstly. We now aim to stick the the Pdu proteins in the order ABNTU and finally JK. While all this was going on we performed more minipreps and stocked bacterial cultures to keep an updated collection of our created constructs. This was a particular highlight for many of the team as it was there first time using liquid nitrogen.
After successfully cloning PduD into pUNI-PROM Brian and Jane began working on GFP and mCherry two of the proteins which will be targeted to the microcompartment by ligating them to pUNI-PROM along with the PduD signal sequence. As these proteins fluoresce this will allow visualisation of protein targeting to the microcomaprtment. The remaining members of the team made use of some of the iGEM biobricks as well as amplifying specific genes from E.coli genomic DNA. More details of this to follow.
Fail of the Week = Kasia’s gel extraction and Rachelle’s pyrotechnics.
Diary: Week 5
After further revision of our cloning strategy we decided to revise the cloning order once more and clone in the order ABTUNJK. The difficulty seems to be arising from the PduN. As it is such a small fragment it is proving difficult to see it on a gel. The team finalised what proteins they wanted to target to the microcompartment. The plan is use the microcompartment to produce lemonade, using limonene and aspartate biobricks from the iGEM directory. Additionally we plan to target arsenic binding protein to use as a potential water purification system and ferritin in order to make our bacteria magnetic. Cloning of these proteins into the PduD20 and 40 is well under way.
As an aside half of the team took part in fire extinguisher training. The remainder of the team will under take this course in the coming weeks.
Diary: Week 6
This week was much the same as the previous week with the team working on individual projects as well as the microcompartment. We also had a team night out which was a great success. In an attempt to get the microcompartment much faster we ordered primers to perform a Gibson Assembly. This would allow complete amplification and formation of our microcompartment in one step.
While the scientists were hard at work our two brilliant computer scientists have released apps for both Android and IPhone. Both apps are now available on the app store and are essential tools for any synthetic biologist. Get downloading people!
Diary: Week 7
The final push is on. Work is still under way to complete the microcompartment as well as individual projects. We have also began cloning of a variant GFP with a SSRA tag attached which targets GFP to CLP a protease sometimes referred to as CLPA/CLPAX within the bacterial cytoplasm. Our hypothesis is that by ligating this GFP to our signalling sequences either PduD20/40 it will be translocated to the microcopartment before it is degraded. This will also allow us to specifically label only the microcompartments.
Work into the Human Practices element of iGEM is also well under way. We have been in contact with the World Debating competition which is being held in Dundee this year and we hope that we may be given a slot where we can perhaps carry out workshops or have a discussion about what synthetic biology involves as well as what we are currently doing on our project. We have also been in contact with Café Science a monthly informal talk followed by a discussion by a scientist about their research area at a local café Chambers. We are hoping that we may be given a slot in the coming weeks.
Diary: Week 8
The microcompartment is getting very close to completion we have now successfully cloned ABTUN into our plasmid and hopefully we will have JK into the plasmid at the end of the week. As for our Human Practices element of iGEM we will be collaborating with the team from St Andrews at the World Debating competition next week. We plan to present both the pros and cons of synthetic biology which will then be followed by an informal discussion into the various issues associated with synthetic biology.
While work on the microcompartment continues the rest of team began cloning their individual projects onto the biobrick plasmid. As well as this an expression strain of E.coli MG165 were transformed with our plasmids so that we may carry out experiments allowing us to fully characterise what we have cloned.
Diary: Week 9
The seven genes which encode the bacterial microcompartment have successfully all been cloned into E.coli and have been fully sequenced. The focus of this week was to make all our bacteria with the various targeting proteins such as GFP/mCherry and ferrtin competent so that they may be transformed with plasmids which have the microcompartment genes.
On Sunday we took part in our discussion along with the iGEM team from St Andrews with participants from the World Debating Competition. This event was a great success with some excellent input from all that took part. Videos will be available soon. Towards the end of the week we all went out on group outing termed the ‘Last Supper’ to a local Italian restaurant which serves the best Italian around many thanks to our supervisor Frank Sargent for taking us there.
Diary: Week 10
The final week of iGEM is upon us. The majority of this week was spent performing Western Blotts and protein purifications as well some 35S radioactive labelling experiments which have provided some very interesting results which will be spoken about in Amsterdam. iGEM has been a brilliant experience and I think I speak for the whole team when I say it is something that if you have the opportunity to do you should definitely take part. The competition provides a great deal of lab experience something which is much sought after but also allows you to take part in a new and very exciting field of biology.