IGEM:IMPERIAL/2009/201009

Dear all, when editing please use comment with name under each section. Feedback will be greatly appreciated. DK & JF

THE PRESENTATION SCRIPT

30 Slides:
2 = title page, team
5 = intro
15 = platform, modules, results etc
5 = applications
3 = summary, ethics, achievements

Slide 1 (Title slide)

• Include: E.ncapsulator text, imperial logo, encapsulator logo
• Hold up pills in tube JF & DK:
o ‘This pill contains x million cells’
o ‘Each cell contains x amount of protein’
o ‘With my pill, I can treat the genetic disease Phenylketonuria’
o ‘And mine is 6 times as powerful as morphine…’

• ‘Welcome to Imperial’s 2009 project: The Encapsulator’.

Slide 2 (Team slide)

• Include: Team picture, advisor pictures, student disciplines (2 Biologists, 2 Biochemists, 4 Bioengineers)
• The 2009 Imperial Team consists of an equal mix of Engineers and Life scientists and of course a number of advisors.

Slide 3 (Problem slide)

o Introduce the problem - why are we doing this project?!
o Theoretical advantages: Diversity, Bioactivity, Biosynthesis
o Projected annual sales in 2010 = $52.2 billion
o Bottleneck: Oral availability
o Use example to illustrate: Insulin discovered over 80 years ago but still using injections.

Royah Vaezi - Would like to know more about the first bullet point, e.g. the actual script.

• Totally agree with Royah, think you should really set out problem, highlight current big issues for protein drugs. e.g. Proetin drugs delivered to intesine, e.g.x(any good examples), suffer from these problems...we want to solve this

Slide 4 (Problem slide 2 - problem drilled down)

• (Get an equivalent to the computer transistor diagram)
o All orally delivered polypeptides share a common predicament: the stomach.
o The stomach is a protease rich acid bath which serves to cut polypeptides into small, non functional pieces.
o If you can bypass the stomach, this will dramatically increase oral availability.
o While protein engineering requires precise knowledge can be used to confer acid resistance, it requires a reductionist understanding of an individual polypeptide. Decoupling this problem from the protein facilitates the possibility of a universal solution.
o certain polypeptides can be modified to enhance acid resistance, it requires an indepth knowledge of the physical
o However, the Imperial team have come up with a solution. The E.ncapsulator is a versatile manufacturing and delivery platform by which therapeutics can ferried through the stomach.
o By decoupling the physical structure of the protein from the problem,

Slide 5 (Mission statement - so they know exactly what we are doing)

• Manufacture a pill with the scope to contain any polypeptide
• And successfully delivery this polypeptide past the stomach.

James - i think it is good to really make the point of our motivtions to make the project reusable - firstly for the "protein production" for different applications and also so the modules can be reused for alt. applications to our proeject e.g. cell-death, encapsulation

Slide 6 (Specs)

• Polypeptide Production
o Specifications: To produce any polypeptide.
o Solution: Proteins can be genetically encoded - logical to use biosynthesis over chemical synthesis
• Polypeptide Protection
o Specification: A) Against stomach acid of pH 1-2 and released past the stomach. Non toxic.
o B) Against dehydration
o Solution: A) Acid resistant capsule - also means we don’t have to purify protein product. Based on natural sources of acid resistance, Lactobacillus, E.coli and B.subtilis were shortlisted as potential chassis. E.coli was deemed the most suitable - possesses a broad range of acid resistance strategies.
o B) Synthesis of a preservative in addition keeping the polypeptide inside the cell is increasingly stable.
• Be safe & socially acceptable
o Specification: A) No risks B) Inanimate
o Solution: Genome deletion - doesn’t damage protein or membrane. Using k12 strain as safe.
• Dosage Control
o Specification: Tunable dosage & robust quality control
o Solution: Macroencapsulation of cells

Slide 7 (Solution Overview Picture)

• Here’s an overview of The Encapsulator system.
• Our E.coli chassis progresses through a series of defined modules culminating in the production of a safe, inanimate pill. This sequential process involves M1, polypeptide production, M2, protective encapsulation and M3, genome deletion.
• Taking this modular approach allows for specialisation (bacteria can devote all it’s energy to the specific task required for that module - increases efficiency).
• However, we’ve taken this 1 step further to make it compatible for industrial production. Integration modules have been designed and incorporated, adding a means of control to this platform.

Royah Vaezi - What are the advantages of the integration modules over a genetic timer? Why was this approach taken? I think this needs to be emphasised as it is the key SynBio aspect of our project.

Slide 8 (Temporal control specs)

- Justify why we use these
• Module 1 specs: Grow up the bacteria as protein production is metabolically demanding and therefore need enough bacteria to carry out the task.
• Solution: Chemoinduction is proven technology and is currently used in industry. The output is instantaneous, long lasting and either on or off at any one time.
• M2 specs: Tuneable delay, synchronous response at a population level.
• Solution: Media defined autoinduction switch - we didn’t use a timer as they are unable to provide a long enough delay, they can fall out of phase and require multiple genes
• M3 specs: Has to be tight (restriction enzymes), responsive to physical cues
• Solution: Thermoinduction, signal can pass through capsule.

Slide 9 (Entire Solution Overview)

• So here’s the final system. We will now tell you the story of our Encapsulator…
• Highlight the chemoinduction part of the diagram to indicate that this will be the starting point.

Slide 10 (Chemoinduction)

• Take a problem & solution approach.

• PROBLEM: While many promoters can process multiple inputs e.g. the original LacI promoter. We wanted a simple switch that was turned on in the presence of IPTG.

• SOLUTION: We chose a modified version of the original LacI promoter that only responds to IPTG. LacI is naturally produced by the cell and represses the promoter but when we add IPTG, LacI is knocked off allowing the expression of our gene of interest.

• Blackbox the genes coding for the polypeptide therapeutics.

• PROBLEM: How much IPTG should be added to maximise output.

• SOLUTION: Two considerations:

1. IPTG is toxic in high concentrations, so we did growth curves with IPTG and found a range that we could work with.

2. We did a dry lab simulation for IPTG input and protein output and then went to the wetlab to conduct the experiment. BUT....the promoter turned out to be non functional! Postulate why this was.

Slide 11 (System Overview)

• Flash up system overview with polypeptide production image circled.

• Mention that we wanted our system to be compatible with ANY polypeptide.

Slide 12 (Polypeptide Production)

• PROBLEM: Our cell can produce any polypeptide that starts with methionine....but not all polypeptides do start with methionine.

• SOLUTION: We developed a linker that can be cleaved by the enzyme enterokinase which is found in the duodenum. Note, I might be able to get some data on this next week to show that it works.

Slide 13 (System Overview)

• Flash up system overview with autoinduction image circled.

• Mention that this is a media defined process.

Slide 14 (Autoinduction)

• PROBLEM: How on earth can you encode information in media!

• SOLUTION: Bacteria naturally sample their media and preferentially consume different carbon sources. Show a model of diauxic growth generated by the dry lab. Then show a picture of the PcstA promoter and say that it is induced by an absence of glucose. Therefore encapsulation only occurs once glucose runs out.

• PROBLEM: Which secondary carbon source can we obtain the best output from.

• SOLUTION: Show the testing construct that we made of PcstA cloned to GFP and show the results from a simple assay that measures fluorescence following overnight culture with different secondary carbon sources.

• Summary: PcstA is heavily repressed by glucose, and in its absence it is best induced by 10% casamino acids. Therefore these results determined the composition of our autoinduction media. This module is highly flexible and could be used for many different applications.

Slide 15 (System Overview)

• Flash up system overview with encapsulation image circled.

• Mention that this involves the production of both an acid resistant capsule and the preservative trehalose.

Slide 16 (Encapsulation)

• Show genetic circuit initially with both sets of genes blackboxed. Mention that we have used the PcstA promoter twice in order to increase modularity.

• Reveal colanic acid blackbox and explain the triple hack in a simple manner. E.g. we needed a safe and tethered colanic acid capsule therefore we used these three genes.

• Show RcsB data: EM, growth curve, packed cell volume, SDS PAGE, pictures of mucoid phenotype. Note, fingers crossed that we get these before the presentation.

• Reveal trehalose blackbox and briefly explain how trehalose is made and what it does.

Slide 17 (System Overview)

• Flash up system overview with thermoinduction image circled.

Slide 18 (Thermoinduction)

• Remind them that thermoinduction controls genome deletion module and was chosen becuase the message can be transduced through the colanic acid capsule.

• Explain the theory of the mutated CI repressor that is temperature sensitive. Mention that the high induction temperature used is not high enough to denature the polypeptide or kill the cell.

• PROBLEM: Restriction enzymes are dangerous, so how tight is this thermoinduction.

• SOLUTION: Show our GFP construct and corresponding thermoinduction results. Also show dry lab modelling results.

• Conclude that we have recharacterised this part and shown that firstly it works and secondly it is suitable for use in our system.

Slide 19 (Genome Deletion)

• Explain the theory of restriction enzymes. E.g. they will chop up DNA but leave cel intact. Say why we chose short base cutters.

• PROBLEM: How can we increase the tightness of our system.

• SOLUTION: We chose two restriction enzymes that work better at higher temperatures. Therefore it is highly compatible with thermoinduction. We chose a DAM positive strain such that the genome has greater protection against leaky expression.

• Show wet lab results for in vitro genomic digest.

Slide 20 (Secondary Encapsulation)

We looked at three materials for secondary encapsulation: Xanthum gum, milk protein & gelatin.

• PROBLEM: Colanic acid is sticky... The final product must be easy to handle.

• RESULT: We found that bacterial pellets could easily be incorporated into all three materials. Show images of pellets. But, xanthum gum was very sticky. Therefore score: Gelatin = 1, Milk Protein = 1, Xanthum Gum = 0.

• PROBLEM: Advantageous if the material boosted acid protection.

• RESULT: We found that gelatin immediatly dissolved while Milk Protein and Xanthum Gum retained their more of their structural integrity. However, it should be noted that the encapsulated cells failed a beta galactosidase test highlighting the need of a colanic acid coating. Final score: Gelatin = 1, Milk Protein = 2, Xanthum Gum = 1.

Conclusion = Milk protein is most suitable secondary encapsulation material.

Slide 21 (Platform Showcasing)

• Mention the problems that are still faced after the stomach e.g. intestinal proteases and say that we engineered PAH accordingly.

Slide 23 (Achievements)

Royah Vaezi - It's certainly coming together. Just watch out on timing as you go through. You don't want to spend too much time at the beginning and have to rush through at the end. Also remember to think about the speed at which you'll be talking as this will determine how much you can say. It may be better to cut down on jargon and still get the point across clearly and concisely, rather than using too many words and running out of time for other things. I'll keep an eye out for further developments. Keep up the good work guys!