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

• IGEM:IMPERIAL/2006/ProjectCalendar/2006-9-4 Events
• Day in the lab
• Project Progress

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 ? the fact that MIT couldn't get it to work
  • In our test of s01656, the aiiA doesn't show any significant activity
• Have we tried to be in touch with other iGEM teams using it ? We asked Cambridge about it but they hadn't tested it at that time. We could ask again
• 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 ?
    • John C have proposed that the overall system should not be changed, i.e. the production and death of the prey and predator should be assume unaffected, however, this needs to be verified

• 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 ?
  • The fullest model so far should be
    • dx/dt=a*x/(a0+x) -b*x*y/(b0+x) -e*x
    • dy/dt=c*x*y/(c0+x*y) -d*y
  • Theoretical study on the completed system without chemostat (i.e. e=0 in above equation) is just finished, more simulations needed to be done to confirm the theoretical proof.

• Do we have simulations using realistic values ?
  • No, we are just using special chosen values to define general region of behaviour and extreme cases to characterise the whole system behaviours.
  • By doing this way, we could easily define the expected behaviour with realistic values, it is not difficult to generate simulations with realistic values any way.
  • However, we also need to try to extract realistic values as soon as possible, but currently this is limited by our lab and testing progress.
• How likely our final construct is to work according to simulations?
  • Without the realisic values, it is not possible to conclude
  • Work still needs to be done to find and characterise region that best fit our interest for the system.
• Other issues on modelling?
  • It will be bonus if we could find the limit cycle of the system and characterise them, however it is far too difficult to achieve.
  • Now the main objective should be theoretical analyse the system, finding sets of parameters which will result in a nice limit cycle that are fairly stable and practical. Thus prove the expected system behaviour through simulations.
  • about the behaviour expected from using chemostat, John C suggested that the washing out of AHL in the medium should not affect the overall system, since the more it washed out, the more it will be produced. This is due to the stationary phase of bacteria population. This needs to be verified though.

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
  • Theoretical modelling is not done at all on wiki, since the system is very complicated to analyse. we need to verify and correct all the mistakes before putting the finalised version on the wiki. or should we just upload them even if they contain errors?

• 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