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Issues regarding the project

1. Finalising the oscillator:

  • Finish ligating the predator cell with an aiiA gene that works
  • We received Dr. Fray's aiiA gene. PCR to add the tag was done yesterday and ligations (3) have to be continued until final predator can be finished. The main issue here is time constraint.
  • At the same time, the final predator has been finished ligating with the parts from the registry - this will be minipreped tomorrow and it has to be checked whether we got the correct sequence as well as if the aiiA gene works
  • The test construct (available from the registry) for aiiA is being tested again this week - the actual test construct we are building ourselves has not been finished ligating yet
  • Need to assess whether the aiiA gene from the registry works (any other methods apart from the testing that is being done at the moment?)!!! If it does, then we would save a whole week !!
  • What is happening with the CRE/Lox part? Has it been finished or when do we expect it to finish?
  • Using the chemostat for the final oscillator: How does it effect the system and thus also the modelling? What parameters might we be able to control and does degradation rate change due to washout?
    • The washout will increace degredation rate almost to the point where washout>>>degredation rate.
    • Wash in of nutriants will also affect production rates of all proteins The rate will be directly proportional to growth rate. Therefore If a system in a chemostat tends towards steady state then the flow rate will not affect the final concentration

2. Testing:

  • T9002 does not give good results - do we want to continue any testing with newly electroporated T9002?
  • J37016 gives very good results - do we want to use that as calibration curve instead? YES!
  • J37020 does not work - since J37016 works, we will not continue to test or ligate the monocistronic predator but use the polycistronic predator instead
  • S01656 will be tested again using J37016 in order to see whether aiiA works (Should the protocol be further modified?)
  • Testing the prey cell - How is it being tested and how much has been tested already. When can we expect any results?
  • 4 methods of possibly controlling positive feedback of prey cell:
  • Dilution
  • Riboswitch
  • CRE/Lox - Decided not to carry on with this since there is no chance to finish it off in the next 2 weeks. Nevertheless, the part itself is a great contribution to the registry. Kirsten was working on it too - we will ask her if she wants to continue
  • Using acylase to break down extracellular AHL
  • Which ones are we using - which ones did not work when testing (where are the results from any testing?) and what are the plans for these possibilities?
  • Generally, the data from the testing has to be analysed more toroughly !!!
    • PRIORITY: Need to test J37022, extract the parameters from it and put them into the model!

3. Modelling:

  • There is quite some more work to be done on the modelling but it has been neglected the past days because of labwork
  • Data from the testing should be analysed with the modelling as well in order to see how it affects the system !!
  • Want to understand about the system being used in the chemostat - using the models & how parameters can be affected
  • Need to extract V and K from J37016 and to find out all the remaining parameters

4. Biosensor:

  • Do we want to continue testing ? The last test did not work due to several external issues, should it be repeated at least once in order to see whether it might work ?
  • We are not continuing with the biosensor testing any more. However, we need to assess the activity of the enzyme & carry out experiments to assess the activity (for the use in the final system)

5. wiki:

  • Is it up to date on different issues?
  • Plans have been made to restructure the organization of the wiki but there has not been time to implement it yet

6. Miscellaneous:

  • If we are short of manpower - is there anything we can as John S. and Tom to help us (e.g. data analysis etc.) ? What exactly?