Present Hybrid Promoter Designs cartoon fashion (3 major different types)
Davidson XOR Biological Design
List of auto-inducers and their catalog numbers.
Here is an idea Malcolm and Laurie developed.
![XOR AMC1b.jpg](/GcatWiki/images/5/54/XOR_AMC1b.jpg)
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone (3OC6HSL)}. 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
Oligos_to_Build: Sequences we will need to make this XOR gate.
LIST OF Km Values
- Km of 3OC12 for LasR is 1 nM (Egland and Greenberg, 2000)
- Km of 3OC6 for LuxR is 100 nM (Urbanowski et al., 2004)
- Km of LasR* for operator/promoter is 11 pM (Schuster et al, 2004)
- Km of LuxR* for operator/promoter is 10 nM (Kaplan and Greenberg, 1987)
- Km of cI dimer for OR1 and OR2 is 10 pM (King et al., 1993)
- Km of Mnt tetramer for binding to half operator/promoter is 50 nM and binding whole operator is 1 nM (Berggrun and Sauer, 2001)
- Km of Lsr for its binding site is X nM (no data available)
- Km of AI-2 for LsrR is X nM (no data available)
- Km of IPTG for LacI is 1.3 µM (Gilbert and Muller-Hill, 1966)
- Km of LacI for its binding site is 10 pM (Gilbert and Muller-Hill, 1967)
- Km of LacI-I12 for its binding site is 0.13 pM (calculated from Schmitz and Galas, 1980; and Schmitz et al., 1978)
- Km of LacI-X86 for its binding site is 0.13 pM (calculated from Schmitz and Galas, 1980; and Schmitz et al., 1978)
- Km of LacI-I12_X86 for its binding site is 0.001 pM (calculated from Schmitz and Galas, 1980; and Schmitz et al., 1978)
- Half life of molecules in E. coli: 2 minutes for mRNA; 1 molecule is 1 nM concentration (Fang Ph.D. Thesis, 2007); translation rate 15 amino acids per second and transcription is 40 nt per second (from Genes VII by Lewin).
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 be weak)
2. order of coding sequences (eg. enzymes could be second for lower expression level)
3. identity of repressors (Davidson is building three different LacI repressors)
4. type of degradation tag on proteins
Things to Do
1. make a list of the parts needed and their building status
2. design, order, and clone 4 hybrid promoters
3. test the 4 hybrid promoters with GFP or RFP outputs
DNA Sequences of Relevant Parts
Oligo design for XOR Hybrid Promoters
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.