Notes: I have included all four promoters (the R#### parts), but we can just use one or two if we can decide which promoters we don't want. Also, if the FACS appointment is set up Thursday, then that means someone has to grow liquid cultures late in the day on July 4th. The solution is to switch the FACS to Friday and use Thursday to test the transformed cells. Also, the I5311 YFP control looks green under the fluorescent scope in the large room.
- 7/02/07 Monday
- receive OHHL from Sigma, resuspend, and put in freezer
- send T9002 and J23039 for sequencing
- S03623 and S036308 with EcoR1 and Spe1
- J37034 (we need four samples of this) with Xba1 and Pst1
- B0015 (we need two samples of this) with EcoR1 and Xba1
- R0040, R0051, and R0052 with Spe1 and Pst1
- Set up FACS appointment for Thursday afternoon
- 7/03/07 Tuesday
- Cip the B0015, R0040, R0051, R0011, R0052 digests
- Gel extract all the digests that were done on Monday
- S03608+B0015, S03623+B0015, [R0040, R0051, R0011, R0052]+J37034
- Transform the ligated plasmids into BL21
- 7/04/07 Wednesday
- 4th of July!
- grow liquid cultures of T9002, I13263, I13522, I5311 in preparation for the FACS on 7/05/07
- grow liquid cultures of the transformed ligated parts in preparation for miniprep and sequencing on 7/05/07
- 7/05/07 Thursday
- FACS with T9002 (tetR promoter, GFP), I13263 (GFP control), I13522 (pL promoter, YFP), I5311 (YFP control)
- miniprep the transformed ligated liquid culture samples prepared on 7/04/07
- prepare the miniprepped, transformed, ligated liquid culture samples prepared on 7/04/07 and send for sequencing
- 7/06/07 Friday
- Find some way to test whether our signal sender constructs actually work. One possibility is to grow up the [promoter]+J37034 and see if there is fluorescence. Are there any ways to detect if our signal senders are creating OHHL?
- Find out what kind of access we have to HSL proteins so we can test the response of the receiver construct at different levels of HSL concentration.
- Transform the signal and receiver into two OmpA+His constructs. Mix these two constructs with large nickel beads as a test
- Do something similar to the above idea, except with other OmpA+something constructs
Things to Figure Out
- Read papers and figure out what people did to control the levels of expression of LuxI/LuxR
- What promoter should we stick in front of LuxI?
See the Quorum Sensing protocol page.
Additional Ideas (6/26/07)
This is a pretty awesome paper: Constructuion and engineering of positive feedback loops
- Mentions making LuxR mutants to make them hypersensitive, which would minimize crosstalk ... directed evolution could also be potentially used to enhance the activities of LuxR or alter specificity for a cognate signal molecule.
Initial Plan (6/25/07)
Basically all of the parts that are available are derived from the LuxI/LuxR system found in V. fishcheri. The Voigt paper that we read a long time ago used this system to demonstrate a cell-density dependent expression of invasin in E. Coli (Media:Voigt.pdf).
The basic idea that Perry and I discussed requires two basic parts which Perry transformed into three separate tubes of Top10 cells this afternoon:
The only difference between the two signal receivers is a different YFP reporter protein.
A quick rundown of how it will work: we will stick some sort of promoter in front of the BBa_F1610 in order to produce constitutive expression of HSL. We can play around with which promoter we want to use in order to tweak the sensitivity of the system. This HSL will normally diffuse quickly. Meanwhile, there is constitutive expression of the protein produced after transcription and translation of luxR (called R from now on) is continually going on in the bacteria with the receiver. The HSL will bind to the R and these bound complexes will dimerize and activate transcription of the YFP reporter. However, normally the concentration of HSL is too low and the equilibrium highly favors unbound HSL and R. In areas of high cell concentration, the concentration of HSL will be great enough to shift the equilibrium toward the bound complex. This bound complex will then activate the transcription of the reporter gene. An interesting note is that the bound complex supposedly also represses the luxR gene according to the Biobricks parts list. However, I haven't found any confirmation of this. If it is true, then it means that along with the activation of transcription of the reporter gene the amount of luxR will decrease and transcription of the YFP reporter would probably decrease.
There are a few ways we could approach this:
- Have both the signaller and receiver+reporter parts in the same plasmid
- Have the signaller part in one plasmid and the receiver+reporter part in another plasmid, both in the same bacterium
- Have two different bacteria: one with the signaller part and one with the receiver+reporter part
Perry asked Mike about the first two possibilities and confirmed that both should be doable. The last possibility was demonstrated in the Voigt paper.
I'm not completely sure what the advantages/disadvantages of each system is, but I think we should try all three possibilities.
Also, if we can get this to work, we could potentially build more complex systems that involve logical gates. One example paper (Media:Pulse.pdf) used two bacteria and five separate parts controlled by inducible/repressible promoters two create a pulse of fluorescence.