Team:St Pauls London/Notebook

4th round of meetings - Current work

We are currently doing wet work, as described by our protocols.

We have begun by transforming all of our competent cells with plasmids for some of the parts we will need for our operons:

30th April:

1. Parts F and L as were introduced into competent cells.

Explanation: We did not do everything simultaneously as we had not requested the Kanamycin, Ampicillin and Chloramphenicol plates early enough, but had some Kan+Amp plates left over from the Transformation Efficiency Kit. As parts F and L came on a Kan+Amp-resistant backbone, we were able to start with these first.

1st May:

1. We found the agar plates with parts F and L almost completely dry due to a presumed incubator malfunction.

2. We resuspended parts B, C, J and K. These were chosen as ampicillin plates had been requested for 03/05, and all these parts came on an ampicillin-resistant plasmid backbone.

2nd May:

1. We introduced parts B, C, F, J, K and L into NEB competent cells which we had received with our enzyme kit.

2. A control was also introduced. This was to help determine if the lack of growth was linked to the DNA provided by iGEM, as was the case for one high school team last year.

3. All of the above were left to incubate overnight, as specified in the protocol.

3rd May:

1. We resuspended parts D and E.

2. We introduced parts D and E into NEB competent cells which we had received with our enzyme kit.

3. We introduced 2 negative controls into NEB competent cells to check for growth without modifications.

4. All of the above were left to incubate overnight, as specified in the protocol.

5. We checked on the parts introduced into competent cells on 02/05/13; these had all grown in the incubator, indicating that previous failures were entirely due to incubator malfunctions.

7th May:

1. We checked on the plates which had been incubating throughout the weekend. Our first important part, which composes our entire first operon, had grown on the plate. However, it had been poorly streaked and single, individual colonies were difficult to pick out from the general mass of growth.

2. As there seemed to be some contamination on one of our plates, we re-plated all of the parts in the following way:

  i. All parts found on a Kanamycin-resistant-only plasmid were replated onto 2 separate Kanamycin-containing plates.
 ii. All parts found on an Ampicillin-resistant-only plasmid were replated onto 1 Ampicillin-containing plate.
iii. All parts found on a Kanamycin- and Ampicillin-resistant plasmid were replated onto 2 separate plates (1 "K" & 1 "A").

3rd round of meetings - Science Conference Presentation and Finalising of idea

As the title indicates, we had to prepare a presentation for the Annual Anglo-Japanese Science Conference, held at St Paul’s School. In order to do so, our idea was finalised, and all biobrick parts that we were going to use were chosen. A comprehensive list of these can be found in the “Parts” section of our wiki.

Our system was now as stated in our brief/abstract: a semi-quantitative lactose detection mechanism.

2nd round of meetings - Admin and Research

1. Having settled on an allergen-detection mechanism, we pushed ahead with research regarding the “Ara” proteins, similar to food storage proteins found in many plants, and present on the surface of nuts (in particular peanuts).

Much research was done regarding the breakdown of Ara h 1 and Ara h 2, the two primary allergens present on the surface of peanuts. This research also encompassed the relative binding strengths of these two proteins with immunoassays (ELISAs and other serum-based testing methods).

In the end, the issue was raised that mediating the response via cAMP and a secondary messenger would be more difficult and require a receptor on the bacterial cell surface. This idea was therefore scrapped, as we could not find how the promoter/operons would be acted on by Ara h proteins.

2. Other allergens and molecules to which humans display adverse reactions were investigated. The most convenient of these was lactose.

Dr Shammas had mentioned the lac operon to us a few prior to the peanut allergen idea being scrapped. We therefore thought it convenient to look into this molecule, which we knew caused issues such as lactose intolerance.

3. We continued researching the lac operon, and starting looking at the kinds of things we could do. A vague idea of a traffic light system based on concentrations within the cell was devised.

4. At this point, we also finalised our application to the iGEM competition and paid the entrance fee.

1st round of meetings - Brainstorming

Ideas pitched:

1. “Operation Gloop” - production of a glue-like protein as found in mussels
2. Ionising Radiation detection
3. pH buffer - regulate acidity of soil for improved crop growth
4. Desalination
5. Organophosphate detection and degradation
6. Screening of blood (pathogen-sensing bacteria)
7. Chitinase-producing bacteria
8. Plastic/Artificial Polymer degradation
9. Allergen sensor

Issues raised with these ideas:

1. Protein may require post-translational modification; bacterium may not have necessary organelles
2. No simple detection mechanism
3. No simple mechanism
4. No simple mechanism; in addition, organism may die due to water potential differences
5. Major issue in that even in cleaving the phosphate group from the molecule, the long alkane chains in an of themselves may be toxic; in addition, a chemical breakdown mechanism which is both much faster and much more efficient than anything we could achieve already exists
6. Difficulty in finding specific proteins to trigger a promoter which are also present on bacteria
7. No simple mechanism/no biobrick
8. No simple mechanism; similar to organophosphate problem
9. Nuts may prove difficult, as allergenic proteins may not have a pathway into the cell to affect the promoter itself; secondary messenger system may have to be employed; could get quite messy