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.

Vincent 05:27, 31 August 2007 (EDT)

Specifications

• Be able to produce a visible FP signal within 3 hours: this is property is also heavily influence by the construct you are expressing. It is a chassis that you are trying to specify. Some stuff about that were already discussed, but unfortunately dropped .see chassis
• These specifications imply a further requirement in the method of vesicle production : the number and volume of vesicles produced must be reproducible and reliable.: this is very important, should have the same status as the other specifications. But more than number and volume at the vesicle level, I would define a measure of functional enclosing volume per ml (due to the all population of vesicles), and also a stability of this measure over time and temperature.
• Would be good to relate permeability of the vesicles to a molecular size.

Dirk 07:25, 31 August 2007 (EDT): I have changed the report to reflect a few of your comments. The specs list now have the reproducibility item as well, and a comment about protocols and reproducibility has been added in the 'work done to date' section.

With regard to the 3 hours specification - I understand your point, it is the chassis that needs to be specified. That was left in there (after considering this point) because it is necessary to ensure that the chassis will not interfere with the signal. This relates to the work you pointed out, by James and Lucas.

Relating permeability of the vesicles to a molecular size is either difficult or restrictive - if I choose a cutoff radius (of 7&angstrom;) then this can only be achieved through pore proteins, and then the specifications limit the solution to pore proteins only. (Personally, I have no problem with that - I think pore proteins are a far better solution.) Detergents will not have a clear cutoff radius - they disrupt the membrane temporarily, and it is very difficult to characterise this disruption. I have extended the specifications, by saying that the membrane should be impermeable to the larger molecules. However, I feel this is still a bad spec because I can't see how this would be measured...

What needs to be done to achieve the specification

• Define a robust protocol in order to produce reliably vesicles (with stable enclosing volume, stability and permeability)
  • as describe later, it is best to define some sub-protocols to approach this end goal with a incremental methodology.
• Define protocols to quantify enclosing volume, stability(time and temperature) and permeability.

Dirk 10:09, 31 August 2007 (EDT): These points are already covered in the Assessment of Function section. I have edited the section to include a statement that protocols need to be defined.

Vesicles containing Cell Extract

• It is tested whether vesicles enclosing cell extract can be produced.: if the expression is not working you will have a hard time to know where it went wrong (is the gene being expressed for example ?). Is it possible to mix GFP with the cell extract ?

Dirk 10:09, 31 August 2007 (EDT): Yes, GFP can be enclosed with the extract. In fact, that was the plan, although at some point we would need to be able to test expression as well. The intention was to include a basal level of GFP, which would provide the control line, and then measure the fluorescence increase on top of this basal line. I have edited the report to make that more explicit.

Work done to date

• links to protocols used (with time and money spent) + feedback on the experience gathered (what went well ? what went wrong ? what could be improved ? ...).
• you have produce vesicles, but what did you observe in terms of the reliability of the process ? Is it achievable to have consistent results ? what are the most sensitive parameters ?

Dirk 10:20, 31 August 2007 (EDT): I have added a link to the implementation page, which discusses protocols, results, experienced gathered, etc. I feel that this is the appropriate way of addressing the first point above, because a discussion would be too extensive to include in this report. What do you think?

As to your second point, we do not have enough data to assess the reliability ofthe process. Nevertheless, I think it is achievable to have consistent results - we just need to find a way to quantify that. There are too many very sensitive parameters in the process, so at this point it is difficult to say which ones are the most sensitive. Here is a list of possible candidates, though: concentration of surfactant, charactersistics of oil, charactersitics (pH, osmolarity) of emulsion solution, quality of dessiccation, quality of suspension mixing, quality of emulsifion mixing, formation of the interface. But there is much more... I don't feel this should be discussed in the article. The reader is pointed to the presentation I gave on the 29th for more information, at the 'Assessment of Function' section.

• Out of curiosity, why have you not used directly the protocol described by Noireaux ?
  • Dirk 07:31, 31 August 2007 (EDT): We did not use directly the protocol described by Noireaux because his protocol was too vague. Instead, we looked up his references, and took the protocol from them. Yet, even then, we did not follow the protocol strictly from the offset because we were advised to use DOPC and mineral oil, instead of the protocol POPC and dodecane. This turned out to be a major cause of lost time for our work.

Next step

• what about the issues related to the solution in which the vesicles are floating ? Do you have any protocols ? Any thoughts about the challenges ?
• Evaluation of the time and money needed.
• It would be good to, at least, draft protocols to assess the function of the vesicles.

Dirk 10:24, 31 August 2007 (EDT): I will address these points in a separate post to this discussion page.

Dirk 10:48, 31 August 2007 (EDT): In response to Vincent's comments on the next step.

• what about the issues related to the solution in which the vesicles are floating ? Do you have any protocols ? Any thoughts about the challenges ?
• Evaluation of the time and money needed.
• It would be good to, at least, draft protocols to assess the function of the vesicles.

Addressing points one and three, no - the challenges have not been looked at yet, so there are no protocols for many of the issues that are anticipated. And, as stated elsewhere, there are no protocols defined for assessing the function of vesicles, yet.

The second point is most important - that of time. In the report, I state the expected delivery dates for each milestone. I didn't go into much detail at each stage, specifically justifying why each step will take one week or two or three, etc. However, should you question the assessment of time required, you can work it out yourself by reading the protocol and the report.

What I would like to see is either a discussion about the repercussions of the conclusions of the report, OR a challenge to the validity of those conclusions.

Way may spend as much time as we want discussing protocols, budgets, methods, challenges, etc. but at the end of the day, we must either accept or reject the conclusions of the report, and then consider the implications. So unless we believe that discussing protcols and challenges will possibly change the conclusions - and I really think it won't - then I don't think we should spend more time on that.

So, we should either challenge the conclusions, or discuss its implications.