IGEM:IMPERIAL/2006/ProjectCalendar/2006-8-18

• IGEM:IMPERIAL/2006/ProjectCalendar/2006-8-18 Events
• Day in the lab
• Project Progress

Report from liaison officers requested by the end of the day`

Assembly

• Which parts are ready and checked ?
  • J37015 complete, maxipreped, sequenced
• Which parts are ready but need to be checked ?
  • J37015RS complete, needs to maxiprep on Monday
  • J37016 complete, maxipreped, sent for sequencing
  • J37019 complete, maxipreped, sent for sequencing
• Which parts are under construction ? When are they expected to be ready ?
  • J37020 = J37031 + J37032 (Polycystronic Predator Test Construct), should be ready on Wednesday (Ligation & Electroporation on Monday)
    • J37031 (ligated & electroporated Friday), miniprep on Sunday, should be ready for maxiprep on Monday
    • J37032 (ligated & electroporated Friday), miniprep on Sunday, should be ready for maxiprep on Monday
  • J37023 AiiA construct with FLAG & LVA tag, will obtain the oligos on Tuesday, so ready for ligation on Friday to make J37024 (aim for maxiprep on Friday)
  • J37024 (AiiA with terminator) see above (Ligation & Electroporation on Friday, Miniprep on Saturday, Maxiprep on Sunday)
  • J37025 (RBS + J37024) (Ligation & Electroporation on Sunday, Miniprep on Monday, Maxiprep on Tuesday)
  • Final Predator Construct - J37019 + J37025 (Ligation & Electroporation on Tuesday, Miniprep on Wednesday, Maxiprep & Sequencing on Thursday)
  • J04500 + CRE - CRE oligo will arrive on Monday, so ready for ligation on Friday (Ligation & Electroporation on Friday, Miniprep on Saturday, Maxiprep on Monday)
  • J04500 + Cre + B0015 (Final CRE Part) (Ligation & Electroporation on Monday, Miniprep on Tuesday, Maxiprep & Sequence on Wednesday)
  • LOX + (C0261+I13504) - LOX oligo will arrive on Monday, so ready for ligation on Friday (Ligation & ELectroporation on Friday, Miniprep on Saturday, Maxiprep on Monday)
  • F2620 + LOX + C0261 + I13504 - (Final LOX Part) (Ligation & Electroporation on Monday, Miniprep on Tuesday, Maxiprep & Sequence on Wednesday)
• Define a priority list for the constructs to be built.
  • AiiA construct is currently the priority
• About aiiA, what if the MIT part is faulty ? have we checked before spending time to attach an immuno-tag ? any back-up plans ?
  • We have a response from the people in Singapore who have researched AiiD (AHL-acyclase). According to their research, AHL-lactonases work much better than AHL-acyclases, so it is not worthwhile using AiiD. They do not have AiiD in purified enzyme form but they do have the sequence. However, we need to get approval from the A-star institute as it is a patented sequence.
  • S01656 (4G) AiiA test construct with LVA tag is ready to be tested on Monday
  • Back up plan: start praying, sometimes money helps
  • We have not heard a response from the AhlD research in Korea.
• What do we want to achieve by the end of next week ?
  • Ligations as described above
• Any other issues?
  • Our ligations will not finish until Thursday of Week 9, assuming that everything goes smoothly.

Testing

• Which parts are ready to be tested ?
  • J37015, J37016, T9002, J37015RS(Next monday), J37020 (Next Wednesday)
• Which protocols are finished and validated?
  • click here for more info.
• Which protocols need further work ?
  • See link above
• For each protocol, do we know what to do with collected data?
  • Still waiting for finalised version of modelling to verify with previous protocols
• What is the status on the chemostat ? What have we learnt ?
  • Nothing on wiki yet after the meeting for chemostat
• What is our current strategy to monitor ratio between our 2 cell populations ?
  • No decision have been made, currently seems to difficult to find an excellent way, click here for more info
• What is our testing strategy for next week ?
  • We are gonna test J37015, J37016, T9002, J37015RS, J37020(completed by next Wed), details to be confirmed.
• Any other issues?
  • No special issues at the moment

Modelling

• What is the current status on the Lotka-Volterra ?
  

Jimmy: 'We're one stage from the final step but it's very complicated - Matthew is helping.'

• Do we have models for each test constructs ? How are we going to use experimental data to get our parameters ?
  

We have completed models for each test construct - an update of all our modelling has/or is being been put up on the wiki as I type. We have yet to look at how to use our experimental data to extract our parameters.

• What is the current status on the full system modelling ?

A full system model has been compiled (it's been sent to Vincent for checking).

• What do we want to achieve by the end of next week ?

By the beginning of next week we should have developed methods for extracting our parameters from our experimental data and developed the spreadsheet into which we can input our experimental data and obtain our parameters as an output.

• Any other issues?

Perhaps we should model our final system on matlab: cell designer doesn't seem to appreciate oscillations.

Documentation

• Have we been able to keep updated the wiki ?
  • Well for the most part the documentation is up to date.
  • The lab notebook has been updated every day, however there are several images of gels from the past couple of weeks that need to be uploaded. This will be done at the weekend.
  • The protocols are being updated on the wiki, hence are always up to date, however the results from the testing of the past two days has not been put up yet - see John C
  • The modelling and biosensor are up to date as of yesterday. Today's progress has not not yet been transferred onto the wiki yet.
• Is there any area of the project which is not enough documented on our wiki ?
  • Most areas are fairly well documented - perhaps something on the chemostat needs to be done.
• Any other issues?
  • Perhaps look at the sandbox (at bottom of main page) - to look at possible alternative colour schemes for our wiki.
  • There is maybe a lack of good linking - we need to look at restructuring some of the information as some pages are quite difficult to locate - for example in "Oscillator parts" there is a huge amount of information, some of which takes 3 or 4 links to get to.

Biosensor

• What is the current status ?
  

Christin and Farah met up with Dr. O'Hare to discuss the biosensor as our original contacts (Professor Cass and Dr. Radomska (Anna) are both away on holiday). He suggested using HPLC as a method for testing AHL concentrations as an alternative to our T9002 assay. Christin emailed Ian Moss at ABC (the ABC handle sequencing and making peptides etc. and are located in the SAF) to request the price of this service and the information they would require e.g. the amount of solution. He replied saying that he wasn't really sure what we were referring to. Perhaps, we should go back to Dr. O'Hare or Dr. Leak (he mentioned there is HPLC equipment available in the bioreactor suite).

Dr. O'Hare also referred us to Suket Singhal. He's a PhD student working on biosensors - in particular, ISFETS. Unfortunately he, too, is on holiday.

As for the standard sensor that we're looking for - Dr. O'Hare mentioned he had 'some lying around'. He said that glass electrodes are probably the best (as did Anna). Glass electrodes are also commonly available for purchase.

• Have we decided to do some practical experiments ?

Our strategy now is to contact Dr. O'Hare in the hope that he could refer us to someone else. Hopefully someone somewhere will be able to guide us on building the sensor, the procedure has been described as 'straightforward'.

The enzyme has already been ordered and it should arrive next week.