Talk:IGEM:IMPERIAL/2007/Projects/In-Veso/Presentations/31-08-07

hey dirk just a few comments -

Specs:

• people don't like the words toxic and hazardous (although we are dealing with hydrocarbons)
• could do with a prioritised list of specifications
• AHL, being a small molecule, maybe able to diffuse in and out of the vesicle (at last thats for E. coli)

Cell by Date
1. Be functional and maintain a constant (or predictable) population for at least 4 days
2. Be functional and maintain a constant (or predictable) population in temperatures between 4°C and 37°C.
3. Be permeable to nutrients required for gene expression

Infector Detector
1. Be permeable to AHL???
2. Be able to produce a visible FP signal within 3 hours

What needs to be done:
- Nice!

Work done to date:

• Pictures?
• empty vesicle formation + use of various lipids and oils

the rest are all fine...

Recommendation (Alex)
In relation to the competition, our team currently has the storyline of detecting stuff, and is showcasing the use of cell-free systems that would allow us to achieve the specifications of our applications, which we were previously unable to due to the limitations of bacteria. Our edge in the competition therefore lies in that we are able to expand the applicative nature of synthetic biology because of our new chassis.

The preliminary results of in vitro systems have so far demonstrated its versatality and its limitations, and not all the specifications required by the applications have been met with in vitro translation. This means that despite the efforts of the team, we are still falling short in terms of the specifications that is required for our applications - something that we are finding solutions to and may not simply due to the inherent characteristics of the system itself.

Where does in veso gene expression come in?

Vesicles are a means whereby we can attempt to circumvent some of the problems that we face with the inherent characteristics of the in vitro system (eg. lifespan). it also provides an alternative platform to in vitro expression in our promotion of cell-free systems. While in vitro systems would no doubt be the main platform for our application, the future of cell-free systems lie in the possiblity of creating an artificial environment where we can control gene expression, and indeed, engineer biology.

The selling point of vesicles

If we choose to pursue vesicle formations, they no doubt will probably not contribute to our applications in time for the jamboree. however with respect to the selling point, and the promotion of the idea of cell free systems as an alternative chassis to bacteria, being able to qualitatively demonstrate the potential functionability of vesicles gives us the oppurtunity to capture the imagination of the audience as we discuss the future of cell free systems. having expanded the realms of applications in synthetic biology, we are not only breaking barriers in terms of application, but are also attempting to pursue a prototype gene expression machine where we can actually design what goes into the machinery, as well as incorporate manufacturing and production methods (like a production assembly).

Im sorry with the recommendations bit. got carried away writing. please edit freely.

cheers, alex

Dirk 18:29, 30 August 2007 (EDT): In reply to Alex's opening comments

Hey.. I'll comment on your comments using the headings you put up. Let's discuss this here, so that we can keep a record of why certain things were said in the report.

Specifications: I can't think of a better way of saying 'not toxic or hazardous'... It is the spec, after all.

I don't know about AHL, so I added in as a separate item. If it is smaller then amino acids, then it should be OK to leave it out. I like leaving it in because it is a specific requirement of ID. That is, if we make our membrane selectively permeable only to ATP and AA's, and it excludes AHL, then it doesn't work even though it meets the spec.

I didn't prioritise because I don't think it adds much to the content of the report itself. What do you think? We can prioritise it. Also, I'm not sure which is best - to leave the specs as one list, or as one for list for each app.

Work done to date:

You're right.. I think I should link up the results, instead of posting them directly. What do you think?

Recommendation:

I like what you wrote.. we should discuss this more..I'll add another comment on this.

Dirk 18:37, 30 August 2007 (EDT): On Alex's recommendation.

The recommendation we make depends on two factors, I think:

1. Whether the end product of our vesicular efforts will be presented at the Jamboree or not
2. Whether it is good use of resources to continue pursuing the vesicle project

(I said some of this earlier when we were discussing in the Caro.) Addressing the first issue, about the presentation - I don't think it should be presented unless it can be tightly integrated with the applications. I don't think it will be good to say that we tried it but didn't have time. Also, if we decide just to add it at the end as a "it's the future of CFS" then I don't see how our continuing to work in this will add to this statement. Sure, it's the future of CFS, but we don't need to start doing it just to say it's the future.

WRT to the second issue, it depends on what the group thinks about how much work still needs to be done in other parts of the project. I'm sure nobody will say that they can spare people. And I'm also pretty sure that if we say that we can help them out, they will be very happy. So the first resource is people, and we can allocate this better as it stands. The other one is money, which I don't go into very much in the report (partly because Sigma doesn't give me some prices), but it will be an expensive pursuit if we carry on.

Cheuk Ka Tong 20:50, 30 August 2007 (EDT) Hey Dirk, looking at the time frame we are working with, I agree that it is unlikely that we will generate in veso data relevant to the Cell By Date and Infector Detector applications. However, I feel that we should not abort the vesicles project in light of the following reasons.

• We are using simple DNA constructs, what makes our project stand out in the competition is our cell-free chassis. By extracting the gene-expression machinery from E.coli and putting this machinery into vesicles, we are effectively making 'artificial cells'. This ties in very well with the fundamental idea of synthetic biology - the engineering of living systems.

• I agree with Alex that demonstrating the functional potential of vesicles will capture the imagination of the audience, so it would be good if we can make selectively permeable vesicles in which gene expression can take place. Instead of citing literature, I believe that if we can showcase glowing vesicles at the Jamboree, the public will be more convinced of the future of cell-free systems.

Ben Yi Tew: I do agree that vesicles tie in strongly with the idea of synthetic biology, and it will be very impressive to the judges and everyone at MIT. But like what the professors have been emphasizing about, a complex system that doesn't work is not as good as a simple, but working system.

I would personally be impressed if we could have gene expression within a vesicle. That would truely mark it as the beginning of a new chassis in synthetic biology. Even if the expression lasted for 2 hours only, that'd be a great start. However, just remember how much difficulty the paper had getting E. coli genes expressed in there.

My point is, I disagree with Dirk in that vesicles must be tied in strongly with the applications to be presentable at the Jamboree. I think getting experiments, calibrations and all that in veso will not be necessary. If we have one or two experiments showing that in veso works with our constructs, I think it would be enough to make a small portion of the presentation. Anyway, you mentioned there is a "chance" of getting expression within the vesicle. I think you should judge whether or not that chance can outweigh their opportunity costs. If you think it's possible, I see no point to stop working on vesicles.