• Design
• Ideas
• Sensor
• Responder
• Switch
• Lysis
• Reference

Responder

Recently the recombinant phage containing genetically engineered DNA has been a new weapon for clinical treatment. This combination enables the extension of phage's innate phenotypes, provide a delivery way for sRNA cassettes. The M13 phagemid/helper system has been widely used in phage display, and been commercialized already. We choose the M13 phagemid/helper system packaging the CAT-targeted sRNA cassettes as our responder.

In our experiment, the M13 phagemid is constructed by the sRNA cassettes and a phage-derived f1 origin of replication. And the helper phagemid is M13K07, coding all the machinery genes required to produce viral particles. When the phagemid and helper plasmid are co-transformed, the sRNA can be packaged into the particles, then the engineered M13 phage is able to infect the live E. coli via the F pilus [2].

The sRNA cassettes combine a scaffold sequence with a 24 base pairs antisense target-binding sequence which can silence the target gene and lead the gene can’t express its product[3]. The scaffold sequence contains an Hfq-binding motif. Hfq protein helps the combination of mRNA target and the sRNA.

As it is said in our project background, we choose acc(3)-Id gene as our target gene. It confers confers resistance to gentamicin, sismicin,and fortimicin. we should have designed our sRNA according to this gene, and test the silencing effect of the sRNA on E. coli 0157:H7 which contains acc(3)-Id gene. However, E. coli 0157:H7 is a kind of very dangerous Pathogenic bacteria, we can use it in our experiment considering the safety. So we choose CAT gene in the harmless E. coli as the replacement of acc(3)-Id gene.

The CAT gene leads the CAT resistance in bacteria. This CAT-targeted sRNA cassettes are competent to knocking down the expression of CAT gene, when joint used with certain antibiotics, the CAT resistant bacteria can be killed.

Switch

Figure 6. The diagram of the mechanism that how the switch consisting of the cI gene and lacI gene works.

When the signaling molecules reach a certain level, the sensor will be activated and stimulate the responder. In order to have a better efficiency, we used a lysis gene to crack the cell for the responder releasing. And for a larger repression of the responder, we used the toggle switch to provide more time before the lysis [4].

Our switch contains the promoter Regulatory lacI+PL (BBa_R0011), lacI gene (BBa_I732820), Pr promoter (BBa_R0051) and cI gene (BBa_P0451). The LacR protein produced by lacI gene can inhibit Regulatory lacI+Pl promoter, the CI protein produced by cI gene can inhibit the Pr promoter [3]. The lysis consists of Pr promoter and lysis gene (BBa_K112806.

When the concentration of AHL around the engineered bacteria in a low level, AHL cannot be sensed by the Lux sensor part, so the responder and lacI-1 gene is inactivated, and the Regulatory lacI+PL is able to stay an activated mode. It leads an expression of cI gene at its wild-type level, which inhibits the lysis gene. However, when the AHL molecules reaches a certain concentration, AHL can be sensed by the sensor part, then Regulatory lacI+PL promoter can be activated for the expression of responder and lacI-1 [5]. The products of the lac-1 inhibit the Regulatory lacI+PL promoter so the expression of cI gene is inhibited. As a result, inhibition of Pr promoter is relieved [6]and lacI-2 and lysis gene are expressed. The product of lacI-2 gene can further repress cI gene, and the products of lysis gene make the cell cracked.

Lysis

Our lysis consists of a Pr promoter (BBa_R0051) and a lysis gene (BBa_K112806), this gene can express T4 endolysin lysozyme, which is from enterobacteria phage T4 degrades peptidoglycan layer. Endolysins are proteins which can help the phage through the bacterial cell. The endolysins can cause lysis by degrading the peptidoglycan, which is a necessary substance for the cell wall [7].

When the pathogens reach a certain concentration, the sensor part responds to their signaling molecules and start the transcription of the M13K07 packaging. Then the toggle switch transforms from the “ON” state to the “OFF” state. This transformation needs some time, so enough time is left for the M13 phages to be synthesized. When the switch is in the “OFF” state, the repression of lysis is relieved. The M13 phages packaged and the cell of the engineered bacterium begins to fracture, and the M13 phage is released to kill the target bacteria.

The following picture shows how our gene network senses the signaling molecules AHL and finally release the M13 phagemid containing sRNA cassettes in the programmable bacterium.

Figure 8. Diagram of how the entire gene network works in the programmable cell.

Reference

[1] http://parts.igem.org/Lux
[2] Sagona, A. P., Grigonyte, A. M., MacDonald, P. R. & Jaramillo, A. Genetically modified bacteriophages. Integr. Biol. 8, 465–474(2016).
[3] Bernheim, A. G., Libis, V. K., Lindner, A. B. & Wintermute, E. H. Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli. J. Vis. Exp. 109, 1–10(2016).
[4] https://2013.igem.org/Team:TU-Delft
[5] Kobayashi, H., Kærn, M. & Araki, M. et al. Programmable cells: Interfacing natural and engineered gene networks. PNAS 101, 8414-8419(2004).
[6] Anesiadis, N., Cluett, W. R. & Mahadevan, R. Dynamic metabolic engineering for increasing bioprocess productivity. Metab. Eng. 10, 255-266(2008).
[7] Nelson, D. C., Schmelcher, M. & Rodriguez-Rubio, L. et al. Endolysins as Antimicrobials. Adv. Virus Res. 83, 299-365(2012).