Difference between revisions of "Divide and Conquer Biological Challenges"

Revision as of 20:20, 12 June 2008

* We are interested in building the pLac_lux promoter.

If LuxR and LacI^Q are always on.....when you have IPTG AND NOT AHL


Temperatures and cycles:
95 degrees for 5 min.
95 degrees for 30 sec.
29 degrees for 30 sec
72 degrees for 5 sec.
Go back to step 2 29x

100 ng of DNA was in the PCR (Is this supposed to say 10 ug? -TE)

* We need to get the Lux/AHL system working.

• LuxI
  
• LuxR
  
• lux pR
  
• hybrid plac-lux promoter
  

* We might need to get the Lsr/AI-2 system working.

Our summary of the Lsr system: Davidson/Missouri_Western_iGEM2008#Lsr_.28AI-2.29_cell_signaling_system

• There are NO Lsr parts in the registry:
  
• We will need to find information on the Lsr system:
  

1. We need to find where the LsrA and LsrR promoters are located. We know that they must be located in the region between the LsrR and LsrA genes (1599514..1601049 E. coli K-12 Gene) but we cannot find any papers that describe specific sequences for these promoters. The sequences are unavailable through the MG1655 sequencing center because the site does not keep track of promotor sequences. The cooridantes for the promoters are also unavailable for this strain.
2. We know that CAP binds to the LsrA promoter.

• We will need to make BB parts for:
  

1. LsrR and LsrK which are adjacent to each other in the E. coli genome. We could amplify them together with a total size of over 2600bp.
2. Lsr promoter
3. LuxS that produces DPD that somehow is converted to R-THMF.
4. How do we get cells to make AI-2?

* We do NOT need to use additional chemical input signals (e.g., aTc and IPTG). We will use only AHL and AI-2 added exogenously.

* We need to have an XOR logic gate produced.

Existing Logic Gates

• AND:
  

Existing But Complex AND

Simple But Hypothetical AND
We would need to have:

1. Starts with pTetR
2. over production of LacI^Q repressor but note this link is plain LacI
3. over production of TetR repressor
   
4. Therefore, we will have to build 1) modified promoter and 2) LacI^Q

• NAND:
  

1. I732914

• OR:
  

Simple but Hypothetical OR

• XOR:
  

See ETH's Designed but not build XOR gate. They also published this here
Also, there is a tunable quarum sensor here.
Simple but Hypothetical XOR

• NOR:
  

1. I732916
   
2. I732917
   

• NOT (Inverters):
  

1. I732205 (NOT/ Dual-repressed NOR)

Self-Contained Inverters

1. J23040 (AHL-dependent inverter)
   

PoPS Output Inverters

1. Q04121 (lac inverter)
   
2. Q04400 (tet inverter)
   

* We need to figure out how to get cells to communicate in a sequence and not stop growing too soon.

• What if Amp^R is secreted and cells are not Amp^R?
  

This would prevent cells down the chain from responding too soon. Erin and Pallavi have conducted experiments with this for both distance and timing.