This construct was designed to function as an OR logic gate. Our system consists in two different promoters (A3 and LasB) expressing the same reporter gene (GFP). LasB (BBa_R0079) promoter (used by NYMU-Taipei 2009, Northwestern 2011, Tokyo-tech iGEM 2011 and Tsinghua-A 2011) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed Pseudomonas autoinducer (PAI). A3 promoter is activated by P4, a regulatory protein from phage φ29 from Bacillus subtilis. In this way, if our target cell receive either LasR, P4 or both proteins, it will express GFP protein and we will have an OR gate.

To obtain our final construct we required the following Biological Parts:

Obtained from the Registry:

[http://partsregistry.org/Part:BBa_K143001 BBa_K143001 (AmyE 5’)]
[http://partsregistry.org/Part:BBa_K143002 BBa_K143002 (AmyE 3’)]
[http://partsregistry.org/Part:BBa_R0079 BBa_R0079 (LasB)]
[http://partsregistry.org/Part:BBa_B0014 BBa_B0014 (Double Terminator)]

Obtained from Margarita Salas Ph.D.’s Group:

A3 from phage phi29 from plasmid pFRC54.

Omega cassette from plasmid pHP45Ω.

GFP reporter gene

We designed the following primers to add the RBS site to GusA:

GusA

UPPER 5'-3'
PREFIX+RBS+SPACER+GUSA
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta

LOWER 5'-3'
SUFIX+GUSA
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA tcattgtttgcctcc


We also designed the following primers to obtain A3 from phage phi29 from plasmid pFRC54 and Omega cassette from plasmid pHP45Ω:

A3

UPPER 5'-3'
PREFIX+A3
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga 3'

LOWER 5'-3'
SUFIX+A3
5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'


OMEGA CASSETTE

PREFIX+OMEGA CASSETTE(45 bp)
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG CCGGGGATCCGGTGA 3'

SUFIX+OMEGA CASSETTE (44 bp)
5' GTTTCTTCCTGCAGCGGCCGCTACTAGTA CCGGGGATCCGGTGA 3'

This A3-LasB OR team had to build the following constructs

So we could obtain the final product:

What we have up now is:

Please see our wetlab notebook in the clicking the following image:

OR gate Notebook