This unregulated T7 promoter, which has an integrated RBS, has high levels of transcription when the T7 RNA polymerase is present; thus, in order to express BioBricks™ under the control of this promoter, a bacteria carrying a T7 polymerase gene has to be used. For our proof of concept, we needed to express our proteins in large quantities. The two bacteria used to test our three enzymes, Escherichia coli BL21(DE3) and Erwinia amylovora, 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 our final BioBrick™ contains the three genes that code 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), which are quorum sensing autoinducers. The lack of communication caused by the decrease 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, like Erwinia amylovora, 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, hence, 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 be unable 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.

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 disruption of the 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, that catalyzes 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 detachment 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) and form biofilm (decrease of ci-d-GMP).

This aiiA gene comes from Bacillus sp. 240B1. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduced the release of autoinducers, decreased extracellular pectolytic enzyme activities, and attenuated pathogenicity on potato, eggplant, chinese cabbage, carrot, celery, cauliflower, and tobacco. The successful degradation of AHLs makes this gene a great alternative to inhibit several of the virulence factors from Erwinia amylovora. The results given by Dong et al. encouraged the potential applications of this gene in the future, “Our results show that the aiiA gene product inhibits virulence of E. carotovora when expressed in the pathogen” (Dong, et al., 2000).

The protein produced arrests flagellar rotation in a manner similar to that of a clutch, by disengaging motor force-generating elements in cells embedded in the biofilm matrix. The clutch is a simple, rapid, and potentially reversible form of motility control. epsE is sufficient to inhibit motility and does so by arresting flagellar rotation (Blair, K. M., 2008). Though the EPS operon is normally repressed in B. subtilis, it's beneficial for the original copy of the epsE gene to be knocked out if its synthetically expressed. Although many bacterial flagellar assemblies contain proteins that are similar in shape, there is no guarantee that the epsE gene will function correctly in any host cell other than B. subtilis.

This BioBrick™ counts with a T7 promoter (BBa_J64997), RBS (BBa_B0034), gene of interest (N-acyl homoserine lactonase; BBa_K2471013), and two terminators, T1 (BBa_B0010) and T7 (BBa_B0012). It was synthesized thanks to the sponsorship granted by IDT® and it codes for an aiiA enzyme, which catalyzes the degradation of N-acyl-homoserine lactones (AHLs).

This BioBrick counts with a T7 promoter (BBa_J64997), RBS (BBa_B0034), gene of interest (glycosyltransferase family 2 protein; BBa_K2471014) and two terminators, T1 (BBa_B0010) and T7 (BBa_B0012). It codes for the epsE enzyme, that has been suggested to function in a similar way to a molecular clutch, could potentially be used as a controller of bacterial movement, promoting biofilm formation and inhibiting motility.

References

Blair, K. M., Turner, L., Winkelman, J. T., Berg, H. C., & Kearns, D. B. (2008). A molecular clutch disables flagella in the Bacillus subtilis biofilm. science, 320(5883), 1636-1638.

Dong, Y.-H., Xu, J.-L., Li, X.-Z., & Zhang, L.-H. (2000). aiiA, an enzyme that inactivates the acyl-homoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proceedings of the National Academy of Sciences of the United States of America, 97(7), 3526–3531.