Team:VictoriaBC/://2009.igem.org/Team:VictoriaBC/project/nandgate

The logical function "not and" (NAND) is a function that takes two inputs, which may be either 0/false or 1/true, and gives output according to

Using BioBrick parts, it should be possible to make very simple NAND gates out of E. coli cells.

NAND gate:

Output control: The lambda cI-repressed promoter, controlling the expression of RFP.

Input control 1: The ara promoter, controlling the transcription of cI. Instead of a standard ribosome-binding site, there will be a "ribolock", which causes a portion of the mRNA transcript upstream of the RBS to form secondary structure which prevents binding of ribosomes. This will strongly hinder translation until the structure is forced to unfold.

Input control 2: The lac promoter, controlling the expression of a "ribokey", a non-coding sequence complementary to the upstream sequence of the ribolock. 

With this setup, it should be possible to have input values of 0/false for <sugar> absent and 1/true for <sugar> present, and output values of 0/false for RFP absent and 1/true for RFP present. It is true that it is a less elegant solution than that of the 2007 UTSC team, but it may be extensible to any input provided there exists some method of detecting it; signal inverters do exist.

A second use of this project is to test the efficacy of the ribolocks when under the control of a promoter other than tetracycline, and to see what changes to efficiency can be made in ribolocks using available BioBricks. A third use (if Natasha's project doesn't use this idea) is to test the temperature-sensitivity of the ribolocks by culturing key-less ribolocked RFP at different temperatures and observing at what temperature the locks fail to function.

potential sticking points

• Because the lac-controlled ribokey must be present for the ara-controlled cI to be translated, and the ribokey should degrade quickly compared to the mRNA if not being constantly replenished, any leaky transcription from the lac promoter should have reduced consequences on the level of RFP.
• If too much leaky transcription is occuring anyway, then different ribosome binding sites with reduced efficiency could be used. That choice should help to reduce the occurance of leaky transcription.
• The only available lambda cI coding sequence has a signal tag for rapid degradation; it may not stick around long enough for significant repression of RFP. If this is the case, then the promoter and cI from phage 434 could be substituted in, with nothing more than some extra assembly work.

potentially needed BioBricks:

• BBa_I0500 (arabinose-induced promoter, includes araC gene to ensure repression)
• BBa_R0010 (lactose- or IPTG-induced promoter)
• BBa_E1010 (RFP)
• BBa_B0034 (the BioBrick standard RBS)
• BBa_B0030 or BBa_B0032 (RBSs with 0.6 and 0.3 the activity of the standard RBS)
• BBa_B0014 (the BioBrick standard transcription double termination signal)
• BBa_J23066 (the optimal ribokey from Berkeley's 2006 iGEM submission)
• BBa_J23032 (one of the better ribolocks from same)
• BBa_C0051 (lambda cI coding sequence, with a rapid degradation tag appended)
• BBa_R0051 (lambda cI-repressed promoter)
• BBa_C0056 (phage 434 cI, no degradation tag)
• BBa_R0052 (434 cI-repressed promoter)
• BBa_K128005 (18bp sequence which could be prepended to the ribolock, in an attempt to emulate Berkeley's best lock)

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