IGEM:Harvard/2007/Quorum Sensing

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(Week 4)
 
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=Quorum Sensing=
=Quorum Sensing=
== Planned Work ==
== Planned Work ==
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Past plans/archives here: [[http://openwetware.org/wiki/IGEM:Harvard/2007/Quorum_Sensing/archives Archives]]
=== Short Term ===
=== Short Term ===
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Notes: I have included all four promoters (the R#####), 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.
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====Week 4====
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*7/02/07 Monday
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Issue: only one set of wavelengths can be used on our plate reader, so RFP is probably out of the question ...<br>
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**receive OHHL from Sigma, resuspend, and put in freezer
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*7/9: Colony PCR of all constructs/parts from Friday of last week
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**send T9002 and I23039 for sequencing
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**Run E-Gel
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**digests:
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**Grow up liquid cultures of
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***S03623 and S036308 with EcoR1 and Spe1
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***F2620-E0240
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***J37034 (we need four samples of this) with Xba1 and Pst1
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***F2620-I13507
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***B0015 (we need two samples of this) with EcoR1 and Xba1
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***J23039-T9002
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***R0040, R0051, and R0052 with Spe1 and Pst1
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***J37015
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**Set up FACS appointment for Thursday afternoon
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***I13273
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*7/03/07 Tuesday
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***I13263
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**Cip the B0015, R0040, R0051, R0011, R0052
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***T9002
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**Gel extract all the digests that were done on Monday
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***J04450
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**Ligation:
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***I5311
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***S03608+B0015, S03623+B0015, [R0040, R0051, R0011, R0052]+J37034
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***I13522
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**Transform the ligated plasmids into BL21
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***OmpA without lux
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*7/04/07 Wednesday
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*7/10: Plate Reader
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**4th of July!
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**7 samples
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**grow liquid cultures of T9002, I13263, I13522, I5311 in preparation for the FACS the next day
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**triplicates of 2 concentrations of OHHL (10nM, 100nM)
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*7/05/07
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**21 experimental samples
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**FACS with T9002 (tetR promoter, GFP), I13263 (GFP control), I13522 (pL promoter, YFP), I5311 (YFP control)
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**Positive controls
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*7/06/07
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***Constitutive GFP (I13522)
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**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?
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***Constitutive YFP (I5311)
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***Constitutive RFP (J04450)
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**Negative controls
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***OmpA1 without lux
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***Non-induced samples (just one of each; no triplicates)
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*7/11: FACS appointment
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=== Long Term ===
=== Long Term ===
*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.
*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.
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=== Things to Figure Out ===
=== Things to Figure Out ===
*Read papers and figure out what people did to control the levels of expression of LuxI/LuxR
*Read papers and figure out what people did to control the levels of expression of LuxI/LuxR
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*What promoter should we stick in front of LuxI?  
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*What promoter should we stick in front of LuxI?
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== Completed Work ==
== Completed Work ==

Current revision

Contents

Quorum Sensing

Planned Work

Past plans/archives here: [Archives]

Short Term

Week 4

Issue: only one set of wavelengths can be used on our plate reader, so RFP is probably out of the question ...

  • 7/9: Colony PCR of all constructs/parts from Friday of last week
    • Run E-Gel
    • Grow up liquid cultures of
      • F2620-E0240
      • F2620-I13507
      • J23039-T9002
      • J37015
      • I13273
      • I13263
      • T9002
      • J04450
      • I5311
      • I13522
      • OmpA without lux
  • 7/10: Plate Reader
    • 7 samples
    • triplicates of 2 concentrations of OHHL (10nM, 100nM)
    • 21 experimental samples
    • Positive controls
      • Constitutive GFP (I13522)
      • Constitutive YFP (I5311)
      • Constitutive RFP (J04450)
    • Negative controls
      • OmpA1 without lux
      • Non-induced samples (just one of each; no triplicates)
  • 7/11: FACS appointment

Long Term

  • 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?

Completed Work

See the Quorum Sensing protocol page.

Brainstorming

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:

  1. An HSL signal sender (F1610)
  2. An HSL signal receiver attached to a reporter (I13263 or I13272)

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:

  1. Have both the signaller and receiver+reporter parts in the same plasmid
  2. Have the signaller part in one plasmid and the receiver+reporter part in another plasmid, both in the same bacterium
  3. 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.

Readings

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