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.