Basic Parts

This unregulated T7 promoter has high levels of transcription when the T7 RNA polymerase is present; thus, in order to express BioBricks™ under the control of this T7 promoter, a bacteria carrying a T7 polymerase gene has to be used. For our proof of concept, we needed to always express our enzymes in large quantities. Escherichia coli BL21(DE3) and Erwinia amylovora, the two bacteria used to test the three enzymes, both contain the T7 polymerase gene

A Ribosome Binding Site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation. This RBS is based on the Elowitz repressilator. As one of our final BioBricks™ contains the three genes coding for our three enzymes, we added RBS sequences between each to increase translation efficiency.

This gene encodes for the aiiA enzyme, which catalyzes the degradation of N-acyl-homoserine lactones (AHLs), quorum sensing autoinducers. The lack of communication that will result from the decrement of AHLs will result in the inability to sense population density and coordinate the expression of target genes, which in the case of several Gram-negative bacteria, include virulence factors.

This gene encodes for the yhjH enzyme, which contains a EAL domain that catalyzes the reaction of c-di-GMP into GMP. C-di-GMP can serve as a second messenger to inhibit motility and increase biofilm formation and adhesion of bacteria, thus, the expression of this gene can make the bacteria more motile.

This gene encodes for the epsE enzyme, which has been suggested to function in a manner similar to a molecular clutch. If expressed inside a cell it will disengage the flagellum from the motor proteins in the cell membrane, causing the cell to no longer be able to swim effectively. EpsE could potentially be used as a controller of bacterial movement, promoting biofilm formation and inhibiting motility.

A transcriptional terminator consisting of a 64 bp stem-loop. For the construction of our genetic circuitry, an efficient and reliable terminator was needed.

Composite Parts

Final Biobricks

This BioBrick™ contains the necessary genetic circuitry to constitutively express the aiiA gene, that when expressed, results in the production of the aiiA enzyme, and thus, the quenching of quorum sensing thanks to the decrease in AHLs.

This BioBrick™ contains the necessary genetic circuitry to constitutively express the yhjH gene, that when expressed, results in the production of the yhjH enzyme. This will catalyze the reaction of c-di-GMP to GMP, which will inhibit biofilm formation and promote motility.

(This BioBrick™ contains the necessary genetic circuitry to constitutively express the epsE gene, that when expressed, results in the production of the epsE enzyme. This will cause the disengagement of the flagellum from the motor proteins in the cell membrane, promoting biofilm formation and inhibiting motility.

This BioBrick™ contains the necessary genetic circuitry to constitutively express the aiiA, yhjH and epsE genes, that when expressed, results in the production of the three enzymes of the same name. The combined effect of these will cause the cell to be unable to communicate (quorum quenching), move (motility inhibition) form biofilm (decrease of ci-d-GMP).