Difference between revisions of "Designing XOR Gates - two campus approach"

Revision as of 15:19, 13 June 2008

Davidson XOR Biological Design

The idea is to have two mirrored halves of the system. One is regulated by AI-1 and the other by AI-2. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented by Egland and Greenberg

Missouri Western XOR Biological Design 1

These two XOR circuits are designed to complement each other. Each recieves a cell-to-cell signal (AI-1 or AI-2) and a chemical signal (IPTG or AHL) and processes it into a cell-to-cell signal. Colonies that output AI-1 would alternate with colonies that produce AI-2. The input message to be hashed could be encoded by the presence or absence of the chemical signals, which would also alternate.

Design Variables

1. strength on RBS for each of the coding sequences (eg. RBS for enzymes could we weak)

2. order of coding sequences (eg. enzymes could be second for lower expression level)

3. identity of repressors (currently LacI, cI, and Mnt)

Things to Do

1. document DNA binding sites for all the DNA binding proteins involved

2. start designing the four hybrid promoters

3. make a list of the parts needed and their building status

Above - Input of AI-1 or IPTG turns on production of AI-2 by LuxS. Input by both AI-1 and IPTG allows production of the repressors cI and Mnt, which repress both transcription units. LuxR and LacI are constitutively expressed.

Above - Input of AI-2 or aTc turns on production of AI-1 by LuxI. Input by both AI-2 and aTc allows production of the repressors cI and Mnt, which repress both transcription units. LsrK, LsrR and TetR are constitutively expressed.

Missouri Western XOR Biological Design 2

LacI could be the new LacI X86+I12.

Also, the output gene should be LuxI, not LuxS. With LuxS, the second cell has to have the Las system in place of the Lux system.
JB/AMC

The idea here is that there are two different XOR gate clones. One takes input of AI1 and IPTG and outputs AI2. The other takes inputs of AI2 and IPTG and outputs AI2. These two clones could be alternated in a pathway of colonies. -TE/AG

XOR Based on Tryptophan Anabolism and the TrpR Repressor

Missouri Western XOR Biological Design 3

This circuit connects the two transcription units through synthesis of AI-1 and LuxR, which combine to repress both units. It responds to AI-2 and outputs AI-2. An analogous circuit could have input of AI-1 and IPTG and output of AI-1. However, these two could not communicate with each other.

Above - Input of AI-2 or IPTG turns on production of AI-2 by LuxS. Input by both AI-2 and IPTG allows production of LuxI and LuxR, which combine to repress both transcription units. LsrK, LsrR, and LacI are constitutively expressed.

Davidson Ampicillin Communication: time delayed cell growth

The very first colony has a high copy number plasmid that is Amp and Kan resistant (^R). As this colony grows over time, it will digest ampicillin in an increasingly larger circle shown by radiating circles of blue. The subsequent colonies have a lower copy number plasmid and have a promoter-less version of Amp^R in addition to the XOR construct. The Amp^R coding will have either its native RBS or one we give it.

Davidson Growth Layouts

To enhance the unidirectional flow of Amp^R, we could either grow the cells on a slant or create a vertical stack of agar plugs. The thickness of the plug would be determined by the thickness of the plates we pour. This may or may not help with the diffusion of Amp^R but it is easier to do than microfluidics.