CzrA-ArsR AND GATE

HEAVY-METAL AND

This construct was designed to function as a logic gate, an AND to be specific. This is due to the way the CzrA-ArsR promoter was designed. We used a system that sensed heavy metals in Bacillus subtilis, which was originally designed by the iGEM Newcastle team 2009 (Newcastle University iGEM team. 2009. <<https://2009.igem.org/Team:Newcastle/Project#Cadmium_Sensing>>). This system consists of a fused promoter, which includes both ArsR and CzrA binding sites. Arar and CzrA are metal sensing repressors. They both respond to cadmium, however silver, arsenic, or copper induces ArsR and zinc, cobalt, or nickel induces CzrA as well (Moore CM, Helmann JD. Metal ion homeostasis in Bacillus subtilis. Curr Opin Microbiol. 2005 Apr;8(2):188-95.). If we use two different metals, specific for each repressor we will have an AND gate. Besides Newcastle’s 2009 design, we designed two different fused promoters with the same binding sites but in different order to try different combinations that could make the system more efficient.


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


Obtained from the registry:
BBa_K143001 (AmyE 5’)
BBa_K143002 (AmyE 3’)
BBa_E1010 (RFP)
BBa_B0014 (Terminator)
BBa_C0179 (LasR)


Synthesis Products:
CzrA-AsR 99
CzrA-AsR 98
CzrA-AsR 97
RBS-CI


Obtained from Margarita Salas Ph.D.’s Group:
P4 from phage phi29 from plasmid pRMn25.


Omega cassette from plasmid pHP45Ω.

We designed the following primers to add the RBS site to BBa_C0179, BBa_E100 and P4.

LASR_2.0_seq_registry
UPPER 5'-3'
PREFIJO+RBS+ESPACIADOR+LASR
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atggccttggttgac
LOWER 5'-3'
SUFIJO+LASR_
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA ttattagagagtaat

RFP
UPPER 5'-3'
PREFIX+RBS+SPACER+RFP

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA

LOWER 5'-3'
SUFIX+RFP

GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT



P4
PREFIX+RBS+SPACER+P4
UPPER

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGCCTAAAACACAA

SUFIX+P4
LOWER 5'-3'

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CTACACCATACTTTT


This metal AND team had to build the following construct:
To then add the following part:

The other AND (pBad/pXyl) team was in charge of building this part.

So we Could obtain the final product:


What we have up now is:
AmyE 5’_ArsR/CzrA 99
AmyE 5’_ArsR/CzrA 98
AmyE 5’_ArsR/CzrA 97

P4_CI

RFP_Terminator


P4CI_RFP_Terminator


AmyE 5’_CzrA/ArsR 98_P4CI_RFP_Terminator
AmyE 5’_CzrA/ArsR 97_P4CI_RFP_Terminator

We are working hard to obtain this last construct with CzrA/ArsR 99 in order to finish it.

ARABINOSE-XYLOSE AND GATE

SWEET AND

This construct was designed to function as a AND logic gate. This is due to the way the pBad/pXyl promoter was designed. We used a system that sensed l-arabinose in E.coli which was originally designed by Amelia Hardjasa and used by iGEM09_British_Columbia1,2. In the absence of arabinose, the repressor protein AraC (BBa_C0080) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription1. In the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription1. We also used a promoter inducible by xylose that has been designed for high expression in B.subtilis which was originally designed by James Chappell and used by iGEM08_Imperial_College. Xylose does not induce the promoter xylose directly, but requires the transcriptional regulator XylR (BBa_K143036). Our system consists in a fused promoter which includes both AraC and XylR binding sites. AraC and XylR are l-arabinose and xylose sensing, respectively, repressors. In this way, if we use these two inputs, each specific for each repressor we will have an AND gate.
To obtain our final construct we required the following Biological Parts:


Obtained from the Registry:
BBa_K143001 (AmyE 5’)
BBa_K143002 (AmyE 3’)
BBa_E1010 (RFP)
BBa_B0014 (Double Terminator)
BBa_C0080 (AraC)
Synthesis Products:
pBad/pXyl promoter
RBS XylR

Obtained from Margarita Salas Ph.D.’s Group:
A3 from phage phi29 from plasmid pFRC54.

Omega cassette from plasmid pHP45Ω.

We designed the following primers to add the RBS site to BBa_E1010 (RFP) and BBa_C0080 (AraC):
RFP
UPPER 5'-3'
PREFIX+RBS+SPACER+RFP
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA
LOWER 5'-3'
SUFIX+RFP
GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT

ARAC without LVA
UPPER 5'-3'
PREFIX+RBS+SPACER+ARAC
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTGAAGCGCAA
LOWER 5'-3'
SUFIX+ARAC
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CAACTTGACGGCTAC

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 arabinose-xylose AND team had to build the following constructs:






To then add the following part:




The other AND (heavy metals) team was in charge of building this part.

So we could obtain the final product:





What we have up now is:

AmyE 5’ – pBad/pXyl



pVeg – RBS - XylR




Omega cassette – AmyE 3’



RBS – AraC – Omega cassette – AmyE 3’



We are working hard to obtain these last two constructs in order to finish it:

pVeg – RBS – XylR – RBS – AraC – Omega cassette – AmyE 3’


AmyE 5’ – pBad/pXyl – RBS – P4/LasR – RBS – CI – RBS – RFP – Double Terminador

References

[1] Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565.
[2] Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7.