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).

The sequence of the new part synthesized by IDT encodes for the aiiA enzyme, which catalyzes the degradation of N-acyl-homoserine lactones (AHLs). The gene aiiA for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. The successful degradation of AHLs makes this gene a great alternative to inhibit a virulence factor from Erwinia amylovora which affects mainly the rosaceae family. The results given by Dong, 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, Y.-H., 2000)

Cell–cell communication is crucial for the virulence. The new part synthesized by IDT was used mainly because it has been tested in different assays. Autoinducer-degrading activity encoded by the cloned aiiA gene of strain A24 was confirmed with an in vivo degradation assay. E. coli DH5α expressing aiiA under the control of the constitutive lac promoter on pME6860 was able to degrade HHL whereas a control culture of DH5α was not.

To test whether a single or several aiiA genes were present in isolates A23 and A24, the 0.8 kb insert of pME6860 was used as a probe in a Southern blot against chromosomal DNA of A23 and A24. Hybridization under non-stringent conditions revealed single bands with six different restriction enzymes used. Both isolates gave identical patterns (data not shown). These results indicate that AHL-degrading activity is encoded in both bacterial isolates by a single aiiA gene.

IDT´s new part, encodes for the epsE enzyme, which has been suggested to function in a manner similar to a molecular clutch. EpsE could potentially be used as a controller of bacterial movement, promoting biofilm formation and inhibiting motility. Though the EPS operon is normally repressed in B. subtilis, it is beneficial for the original copy of epsE gene to be knocked out if EpsE is 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

Biofilms are multicellular aggregates of sessile bacteria encased by an extracellular matrix and are important medically as a source of drug-resistant microbes. Another new part synthesized by IDT was epsE, the main reason to use it was because it also has been tested in different assays. EpsE arrested 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. Furthermore, the mechanism by which EpsE inhibits the flagellum is apparently unrelated to its putative enzymatic activity. (Blair, K. M., 2008)

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 acylhomoserine 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.