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

Introduction

This page briefly explains the specifications and process of the Vesicles branch-project. It describes what was set out to be achieved, and what needs to be done to achieve that. A final recommendation as to whether the branch-project should be continued is presented based on the conclusions outlined.

Specifications

The vesicles we aim to produce are intended to be used in the Cell by Date and Infector Detector systems. As such, the specifications for the vesicles produced is based on the requirements of these two applications.

The vesicles produced must: (not prioritised)

1. Not be toxic or hazardous
2. Be permeable to AHL
3. Be permeable to nutrients required for gene expression
4. Be able to produce a visible FP signal within 3 hours
5. Be functional and maintain a constant (or predictable) population for at least 4 days
6. Be functional and maintain a constant (or predictable) population in temperatures between 4°C and 37°C.
7. The number and volume of vesicles produced must be reproducible and reliable.

Why permeability is necessary: It was found in the literature that vesicles are only one tenth as efficient in protein production if they are impermeable to nutrients. In addition, vesicles are diluted in a solution - they are few and far between. Therefore, having a week and dispersed signal would not match the requirements of either application.

What needs to be done to achieve the specification

To meet these specifications, the following needs to be done:

1. Vesicles need to be produced enclosing cell extract
2. Their membranes need to be made permeable
3. Experiments must be carried out to assess function according to time and temperature

These may be further broken down:

Vesicles containing Cell Extract

Vesicles enclosing cell extract for gene expression must be produced. This is carried out in steps:

1. It is tested whether vesicles enclosing proteins can be produced.
   • Confirmation of this comes through visualisation of GFP inside vesicles.
2. It is tested whether vesicles enclosing cell extract can be produced.
   • Cell extract and purified GFP will be added to the vesicles.
   • Confirmation of expression (and therefore functional enclosure) comes from detecting an increase in fluorescence compared to the basal level.
   • This involves production of cell extract solution, and compensations for osmolarity.

Permeable vesicles

Items 2 and 3 of the specifications above require that the vesicle membrane be made permeable. The steps taken to achieve this are as follows:

1. Through research, identify pore proteins or detergents as possible candidates
   • Toxicity, effect on stability, price, availability, procedures, and effectiveness must all be taken into account.
   • This step can be done in parallel with other production steps.
2. The candidate pore proteins or detergents must be acquired and tested
   • In order to save on costs, this step cannot begin until after cell extract has been enclosed inside vesicles. If this is carried out in parallel, there will likely be much waste of materials before the desired results are obtained.

Assessment of function

This is the most complicated step. Because the production process and end product are highly sensitive to many factors (see presentation), this step can only be carried out after selectively permeable vesicles containing cell extract have been produced.

There are two ways in which assessment can be carried out:

1. By analysing individual vesicles en masse, with normalisation accounting for their polydispersity.
2. By analysing populations of vesicles, and ensuring that all samples are reasonably equal.

The first, requires the use of flow cytometry equipment. The second can be carried out in a fluorometer but, as stated, the samples taken for assessment must be uniform, or fall within a certain range of uniformity. This can be achieved in two different ways:

1. Vesicles are produced as and when needed, and the production method is reliable and reproducible within the required range.
2. Vesicles are produced once, in bulk, and the process for collection of samples for assessment is reliable and reproducible within the required range.

Either way, protocols must be defined for the chosen procedure.

Once a method to assess the function of vesicles is devised, then assessment can begin. This involves:

1. Testing vesicle performance in a range of temperatures
2. Testing vesicle performance as a function of time

The assessment process is very time consuming because it requires careful planning and experimental design, as well as the production of suitable samples. The former can be mostly carried out in parallel with work directed at refining the production method, and can reuse much of the work done by the Experimental Design team. The latter, as stated above, cannot begin before production has been mastered.

Risk

The above outlines the path to be taken to meet the specifications. As any research project, each step has an associated risk that something might not work, and time must be spent modifying the process in order to obtain the desired results. Such events inevitably end up consuming more time and money.

Work done to date

Two people have spent three weeks full time working on vesicles, achieving the following:

• We have produced vesicles enclosing proteins (GFP).
  • However, the protocol used has not been tested yet for reproducibility or the durability of resulting vesicles - which is part of the specification.
• We have identified a possible candidate pore protein and detergent, but more research needs to be done before they are purchased and tested.
  • The pore protein, Magainin 2, is very expensive - £259 per mg.
  • The use of detergents for inducing permeability is very unpredictable, and a very large number of tests will need to be carried out before the right substance, concentration, and conditions can be identified. Moreover, the results of such an investigation would only be valuable if the method of production is reproducible.
• Much of the assessment of function planning has been done by the Experimental Design team, but this work has not yet been adapted to vesicles.

Detailed protocols, experimental results, evaluations, and conclusions can be found in the project implementation page.

The next step

The next steps are enclosing cell extract inside the vesicles, and enabling good permeability of the vesicle membrane.

Considering that:

1. the process of creating vesicles currently takes us two days working in the lab before observations can be made
2. yield problems are anticipated as a result of enclosing cell extract and introducing membrane proteins or detergents

it is expected that these two milestones will not be reached before the end of week 10 of the project - at best. Allocating more people to work on this section of the project is unlikely to expedite the process. However, more workers would reduce the likelihood that the milestones are not reached by then, as they would be able to make a valuable contribution in the research required to identify a viable candidate for permeability, and in solving any other problems that may come up.

Beyond the horizon - Assessment of function

Once permeable vesicles enclosing cell extract have been produced, we can begin assessing their function according to time and temperature. As stated above, this will not begin until after week 10 of the project.

Furthermore, time will need to be spent developing a method to collect uniform samples. As mentioned, this will be either by a reproducible production method, or by bulk production and reproducible collection. It is not unreasonable to expect that this process will take a few more days of lab work.

Therefore, assuming everything is done in the shortest time possible, assessment of function will only begin at the end of week 11 of the project.

Conclusions

• It is almost impossible to meet the specifications before the end of week 10 of the project.
• It is extremely unlikely that the specifications will be met before the start of term in October.
• It is very unlikely that the specifications will be met before the iGEM jamboree in November.

Furthermore,

• If two people continue to work on vesicles until the end of week 10, there is a chance that they will achieve the two milestones of cell extract enclosure and permeability.
• If two people continue to work on vesicles until the end of week 12, there is a small chance we will be ready to begin assessment of function after that.
• Allocating more people to work on vesicles is unlikely to reduce the minimum time required to achieve milestones, but will reduce the likelihood that more time will be required.

Therefore, with respect to the presentation at the jamboree in November:

• It is very unlikely that we will be able to present vesicles data that is relevant to the Cell By Date and Infector Detector applications.

Recommendation

Dirk 19:14, 30 August 2007 (EDT)

Taking the conclusions presented, in addition to:

• the fact that we want to win the competition
• the fact that other areas of the project could use more workers
• the difficulty of smoothly including vesicles in the presentation

my recommendation is to stop working on vesicles (after documentation and wrapping up, of course).

However, if we can be convinced that we can talk about vesicles in the presentation - having only managed to achieve gene expression, with no quantitative data - without detracting from our applications and project, and if the group feels that it is a better use of our limited resources, then we can carry on.

As Prof. Kitney said - projects that win are those that have something that work at the end. If we say that gene expression in-veso (without quantitative measurement) is a 'working project', then I feel this is a change in focus for our project as a whole. This is because we are focusing on applications, ID and CBD - these are our projects, this is what we want to have working at the end. And we can't say we have working applications in-veso if we don't get to test them out.

In sum, I feel that if we present vesicles, it will either fragment the presentation or refocus our project.

Alexander.wong 21:02, 30 August 2007 (EDT)

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 because we have explored something that the rest of iGEM have not explored before. in addition, we are not only expanding the realms of applications in synthetic biology and breaking barriers in terms of application, but we 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). This future is a future of true engineering.

In all, i think that if our selling point is about cell-free systems and the advantages it gives us in meeting our specs, then we should pursue vesicles further. however if the selling point is solely on the applications and a functional product, and that there is no place for the promotion of cell-free systems as our 'edge' in terms of project design and the way we went about implementing the project, then we should drop the idea of vesicles and focus our efforts elsewhere.