IGEM:IMPERIAL/2006/ProjectCalendar/2006-9-4

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(Testing)
(Testing)
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*What lead us to think that the MIT aiiA part is faulty ?
*What lead us to think that the MIT aiiA part is faulty ?
*Have we tried to be in touch with other iGEM teams using it ?  
*Have we tried to be in touch with other iGEM teams using it ?  
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*We have received a new aiiA gene. Can we test it before trying to insert it into our construct ?
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*We have received a new aiiA gene. Can we test it before trying to insert it into our construct? <font color=magenta> Um.. I may be wrong here, but I don't see how we could test the gene without a promotor and terminator- i.e. when it is already in one of our constructs. I guess we could sequence it but will that confirm that it will work? At the moment we are trying to ligate as fast as possible.
*Chemostat
*Chemostat

Revision as of 12:09, 4 September 2006



Progress Report

Assembly

  • Define the status of each test construct (under construction, sequenced, validated, faulty, ready for testing, tested, characterized) -multiple choice possible-
    • Prey cell(J37015): sequenced, ready for testing (has been tested before, testing needs to be repeated)
    • Prey cell with riboswitch(J37015RS): sequenced, ready for testing (has been tested before, testing needs to be repeated
    • Prey cell with Cre: Under construction - at PCR stage still
    • Predator sensing part (monocistronic)(J37020): tested - results are on the results page (BUT: the part does not seem to work)
    • Predator sensing part (polycistronic)(J37016): tested - very good results! (see results page)
    • aiiA test construct(J37022): under construction (ligation failed, should be repeated today) AiiA construct being PCR'd using new AiiA gene. If successful, it will be ligated into T9 overhanging plasmid tomorrow and be electroporated. Next ligation step would be on Thursday. Total ligation steps = 3
    • Full Predator construct(J37036):was ligated yesterday - to be validated by miniprep tomorrow
    • Data from testing has to be documented & interpreted; all raw data & a normalized graph itself is on the results page


  • others ???
  • What is the current priority in terms of assembly process ? Why ?

The highest priority right now in terms of assembly was agreed by the team to be the construction of the finalised predator construct using the recently arrived AiiA gene from Nottigham. If our AiiA gene from the registry does not work, then we do not have any form of functional predator cell hence no chane of an finished oscillator. The team agrees that we should try ligate this part using the new gene so that we can put the entire system together using functional individual components. However, we are very pressed for time. If no ligations fail, the soonest this part would be ready is Thursday week which leaves 2 days for testing.

  • Other issues ?

Testing

  • Define the status of each testing protocol (to be written, to be validated, validated, data collected, data analysed, faulty protocol) -multiple choices possible-
    • Prey cell:
    • Prey cell with riboswitch:
    • Prey cell with Cre:
    • Predator sensing part (mono):
    • Predator sensing part (poly):
    • aiiA test construct:
    • Full system:
  • aiiA from MIT:
  • What lead us to think that the MIT aiiA part is faulty ?
  • Have we tried to be in touch with other iGEM teams using it ?
  • We have received a new aiiA gene. Can we test it before trying to insert it into our construct? Um.. I may be wrong here, but I don't see how we could test the gene without a promotor and terminator- i.e. when it is already in one of our constructs. I guess we could sequence it but will that confirm that it will work? At the moment we are trying to ligate as fast as possible.
  • Chemostat
    • What is the medium to be used in the chemostat ? At which OD do you plan to operate ?
    • Which parts do we plan to test in Chemostat ? Protocols ? Do we know how to get the parameters of the part from the experiment ?
    • Have we done the growth curves of the parts we want to test in the chemostat ?
    • Do we have a final strategy to monitor the ratio between our two populations ?

After speaking to kirsten about this and much discussion amongst the team, we came to the conclusion that once we have a mixed culture, there is no way of controlling individual population sizes. If we wanted to we could use aliquots in order to assess our population sizes but this would be inaccurate too (we would have to grow them up on plates with different antibiotics and we would have to assume the growth rate on plates was a good indication of original pop size in the medium. Also, we would have to engineer in different antibiotic resistance genes into our bacteria to separate them out). The best we can do it measure the OD of individual populations before mixing them and ensuring they are equal. John C says that it is unlikely that the chemostat environment would allow for one species to outperform the other significantly within a short time period.

    • Do we understand how the chemostat environment is going to modify our modelling approach ?
  • Biosensor
    • What is the name of the enzyme we are using ? reference ? see link in Testing Protocol (there are 2 protocols - one for calibration, the other one to check activity of enzyme)
    • Have you already done some testing ? Is there a protocol ? see Testing Protocol (protocol has to be updated). First test was done on Thursday last week, however due to limit in AHL solution being available, the results of the testing might not be accurate
  • How promising is it ? Future work ? After more AHL has arrived, a further test will be carried out and then a better judgement can be made.
  • Data from fluorescence reading has to be interpreted but will probably not give reliable results since T9002 assay itself does not give expected results


  • Other issues on testing ?

Modelling

  • What is the status on the theoretical study of the system ?
  • Do we have simulations using realistic values ?
  • How likely our final construct is to work according to simulations ?
  • Other issues on modelling ?

Doc & Presentation

  • Is the wiki up to date according to the work done during the last two weeks ? Apart from the lab notebook and some things on the biosensor, which are fairly up to date, not a lot has been added to the wiki in the last two weeks
  • Have we started to work on the presentation of the project for the Jamboree ? No, we will probably leave that until we have finished with all the lab-work
  • Other issues on documentation ? Just that some serious restructuring and sorting out will be needed at some stage
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