Final Circuit

For the final Colisweeper circuit we plan a four plasmid system. The mine cells constitutively express LuxI for signal generation and NagZ as identifier hydrolase. In the non-mine cells LuxR is expressed constitutively to process the OHHL signal. PhoA is expressed constitutively as well as reporter for safe cells. Aes and GusA are expressed from pLux promoters with different sensitivities. You can find all the biobricks we used and our own new biobricks in the figure below.

Cloned Constructs

To get to the circuit mentioned above we tested different versions of the circuit. For example we started our experiments using GFP as a reporter instead of the hydrolases. Then we also tested different LuxI and LuxR generating constructs. In the following table we list all the biobricks we used, the plasmids we cloned and what experiments we used them for. In general we used standard biobrick cloning techniques as described in the methods section. Whenever we used PCR gene amplification for cloning, we list the primers used in the following table. To be able to co-transform different plasmids we used backbones with compatible origins of replication and resistance genes. In the table you can find which backbone versions we used for which constructs.

GFP constructs
	Description	Cloning	Maps
1	Receiver cell construct for GFP diffusion experiments	BBa_J09855 backbone (SpeI, PstI) and BBa_E0840 insert (XbaI, PstI)
2	Library of the Receiver cell constructs	Using the BBa_J09855-E0840 construct a library with mutated pLux promoters was created through site-saturation mutagenesis to screen for promoters with changed sensitivities for OHHL:
3	Receiver cell construct for GFP experiments without the LuxR generating part	BBa_R0062 backbone (SpeI, PstI) and BBa_E0840 insert (XbaI, PstI)

LuxI generating constructs
	Description	Cloning	Maps
4	Sender cell construct with a very strong constitutive promoter from the BBa_J23100 promoter library for GFP and Hydrolase experiments	BBa_J23100 backbone (SpeI, PstI) and BBa_K805016 insert (XbaI, PstI)
5	Sender cell construct with an intermediate constitutive promoter from the BBa_J23100 promoter library for GFP and Hydrolase experiments	BBa_J23118 backbone (SpeI, PstI) and BBa_K805016 insert (XbaI, PstI)
6	Sender cell construct with an intermediate constitutive promoter from the BBa_J23100 promoter library for GFP and Hydrolase experiments	BBa_J23110 backbone (SpeI, PstI) and BBa_K805016 insert (XbaI, PstI)
7	Sender cell construct with a weak constitutive promoter from the BBa_J23100 promoter library for GFP and Hydrolase experiments	BBa_J23114 backbone (SpeI, PstI) and BBa_K805016 insert (XbaI, PstI)

LuxR generating constructs
	Description	Cloning	Maps
8	constitutive LuxR generating biobrick	BBa_J09855
9	constitutive LuxR generating biobrick, with negative feedback-loop at high OHHL concentrations	BBa_F2621
10	constitutive LuxR generating construct, repressible through LacI	BBa_R0010 backbone (SpeI, PstI) and BBa_I0462 insert (XbaI, PstI)
11	constitutive LuxR generating construct, with negative feedback-loop at high OHHL concentrations	BBa_R0063 backbone (SpeI,PstI) and BBa_I0462 insert (XbaI, PstI)
12	auto-inducible LuxR generating construct with positive feedback loop	BBa_R0062 backbone (SpeI, PstI) and BBa_I0462 insert (XbaI, PstI)
13	negatively regulated pLuxL-LacI construct to improve leakiness of LuxR system	BBa_R0063 backbone (SpeI, PstI) and BBa_C0012 insert (SpeI,PstI)

We thank our sponsors: