IGEM:IMPERIAL/2009/Feedback & Debriefs/Feedback 24 7

Presentation with Professor Kitney and Advisors

Q : How well characterised are these enzymes (PAH and Cellulase)?

A : cellulase = good, PAH = a few problems that need overcoming, but sequences isolated.

Kirsten: We can always choose other enzymes if these don't work out.

Want to cut down to one application. Want an important application for wow-factor. PKU is a v emotive issue, (good for iGEM!!)

Q : Is there anything clever or new about creating enzymes with a bacteria?
A : If we want to boost yield by whatever amount, then very possibly (using over-expression etc).
How risky is the encapsulation? Feasability in the 10-week timeframe?

Q : Can modules be developed seperately? Are the interactions between the modules important? Or can we decouple seperate modules?
A : yes, very modular. We have tried to develop each module seperately, decoupling each individually from the main project for the moment. Allows development of each, and then the links can be developed further later (timer mechanism for example). Encapsulation is the selling point for our project, we hope to leave the application somewhat generic, at least for the moment. There are many enzymatic diseases, could potentially be applied to any of these, or for delivery of any peptide therapeutic.

Q : What happens when the cell dies? Floating 'sack' of protein?
A : Possibility of using the CaCO3 encapsulation idea before consumption of the bacteria. Will then degrade in the stomach leaving the bacteria protected by the encapsulation. Have also considered using gelatinous capsules, as with other pharmaceuticals.

Q : How big are these capsules? How much will we need to over-express the enzyme production in order to get a good enough yield?
A : Size obviously dependant on application and dosage. initial calculations suggest we need to boost production by about 10 times to give approximately enough enzyme for 1 capsule/meal.
PKU at the moment requires about 8 capsules per meal for amount of enzyme required (to digest 5g av amount of phenylalanine).
Matthieu - good opportunities for some modelling here. How much drug we need to break down phenylalanine before aborption.

Comment: If we can get each module working, then we have a very good project. Working encapsulation is what we really should aim for - the selling point of our project. The endpoint of our project is a 15 minute presentation at MIT; our idea is simple enough to present = good.

Q : What possibilities are there for Biobrick submissions?
A : Several for each module - enzyme production, encapsulation method, killing strategy.

Module 1
Q : How complicated is this module likely to be? (enzyme production)
A : (Kirsten) Not too difficult really, can submit sequence to geneart.

Q : Any advantages of biobricking, rather than just submitting the sequence?
A : Obvious competition advantages for having a good library of biobricks submitted. Other applications for submission of the biobrick.

Module 2
Kirsten : We will probably have to take genes out and use RBS's from e-coli. cannot synthesise genes all in one strip.

Kitney : we have the research project on one hand, but the competition on the other.
Q: How much reliance do we have on Geneart? they are the rate limiting factor for the project, although no other options. When do we need to order?
A : dependant on size of genes, though first biobricks should be submitted at the beginning of next week. Takes time to proofread before submission, and several weeks for geneart to produce the sequences.

Module 3
Q : How straightforward is this module? sounds fairly simple, but is it likely to be so easy?
A : Matthieu : could use already designed biobricks and recharacterise. easy to measure.
possibly not quite as simple in the labs with restriction enzymes. still must overcome problem of basal expression.

Matthieu : Still a bit unconvinced by encapsulation, thinks we are oversimplifying some of the engineering of pathways. May be quite difficult, BUT, if this does work, then we have a great project.

Modelling
Where can the modelling fit into this project?
Killing : We can probably get a lot of good modelling done for this module. we can work backwards from absorption readings.
Encapsulation : No chance! Wont be able to model properly.
Dosage : We can work out some values for the dose control. Preliminary calculations already done, but obviously a lot more needs to be done.

Final Thoughts
- We need to get into the wetlabs ASAP!!
- Covering 2 bases, manufactuing and medicine.
- We need 2-3 people tackling the same problem, gives fresh sets of ears on each.
- We can always decide on an application later on, can keep both ideas running for the moment.