Difference between revisions of "Team:Tec-Chihuahua/Parts"

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 induce expression of this BioBrick™ under the control IPTG will be used.

The pelB leader sequence is a sequence of aminoacids which, when attached to a protein, directs the protein to the bacterial periplasm. Protein secretion can increase the stability of cloned gene products. It was shown that the half-life of the recombinant proinsulin is increased 10-fold when the protein is secreted to the periplasmic space.

Defensin 1 is synthesized in salivary glands and it plays an important role in the social immunity system of the bee, meaning that it is commonly passed through generations. Defensins unpolize inner membranes by causing an ionic outer flow using preexistent channels. Apart from these processes, they are responsible for respiratory inhibition 3 minutes after they are added by directly attacking the respiration chain components.

Antimicrobial peptide that shows activity against both Gram negative and positive bacteria. When being alone, it shows no activity against E. coli, at least at concentrations up to 200 μM. Hymenoptaecin a native peptide showed activity starting at 2μM concentration. When working together, helps by opening channels for the abaecin, which then gets to attack the DnaK, getting to interfere with a proper folding of the proteins.

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.