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<p>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. </p> | <p>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. </p> | ||
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− | + | <p><center><strong>Figure 5.</strong> After the luxpR promoter is activited, the M13 phage is produced and the sRNA cassettes is pakaged into it.</center></p> | |
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Revision as of 13:03, 18 October 2016
Design
Appropriate attribution is half of success.
Ideas
In analogy to electronic circuit, the construction of a programmable cell can be divided into input, engineered gene network, output three modules. The components of each module can be reassembled for the diversify clinical treatments.
Based on this idea, our team designed a gene circuit model and apply it into a certain gene circuit. When putting the circuit into bacteria, the bacterium soldiers form. Our gene circuit contains four parts:sensor, responder, switch, and lysis. The components of the circuit can be disassembled and assembled.
During our brain storm, the gene circuit "Plug and Play" designed by Kobayashi et al. helped us to find the design strategy for constructing the programmable bacteria. The gene circuits of detecting and killing MRSA from iGEM Team TU-Delft 2013 and LMU 2014 also give us revelation. And we used the M13 phage delivery system designed by iGEM Team Paris Bettencourt 2013 as our responder. Finally, we created a gene circuit which can which can combat with the multidrug-resistant bacteria.
![](https://static.igem.org/mediawiki/2016/2/2a/T--XMU-China--Design1_and_.png)
![](https://static.igem.org/mediawiki/igem.org/5/5a/T--XMU-China--Design2_and_version2.0.jpg)
In this model, we aimed to construct a programmable cell which can sense particular biological signals, and later lyse itself for releasing the responder and controlling its population level.
The sensor part can sense the signaling molecules . The responder represents the gene can kill the drug-resistant pathogens itself or combined with antibiotics. The toggle switch is used to control the expression of the lysis gene. When the switch is in the “ON” state, the expression of the lysis gene is repressed. When the switch is in the “OFF” state, the repression of the lysis gene is counteracted.
According to our circuit, we choose the LuxI/LuxR system as our sensor part. The toggle switch contains lacI gene and cI gene. And the responder is M13 engineered phage packaging the sRNA cassettes. Then, a gene circuit which can sense the signaling molecules AHL and silence the antibiotic resistance gene of Enterohemorrhagic Esceriachia coli is constructed.
Here comes the concrete gene circuit.
![](https://static.igem.org/mediawiki/2016/c/c8/T--XMU-China--Design3_and_.png)
Sensor
As mentioned above, our engineered bacteria are designed to detect the signaling molecules and start the responder gene.
Quorum sensing plays an important role in the bacteria communication. Quorum-sensing bacteria can express and release small chemical signal molecules called autoinducers. The concentration of the autoinducers increases when population rises. Bacteria can detect a minimal threshold stimulatory concentration of autoinducers, then lead a relevant gene expression.
One quorum sensing system in Gram-negative bacteria is LuxI-LuxR type. LuxI is the autoinducer synthase to produce the acyl-homoserine lactone (AHL) autoinducer, and LuxR is a protein which can bind AHL autoinducer and then activate transcription. In our sensor, the right hand lux promoter (Lux pR) can be activated by LuxR-AHL component[1].
The sensor consists a promoter (BBa_J23100) and LuxR gene and Lux pR (BBa_A340620). The BBa_J23100 is a strong promoter that can be on in strains without any inducer, so LuxR gene can express constantly. The autoinducer AHL will bind LuxR when the concentration of the AHL up to the threshold. Then the promoter Lux pR will be activated, then the responder start to express.
![](https://static.igem.org/mediawiki/2016/b/b1/T--XMU-China--Design4_and_.png)
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.
![](https://static.igem.org/mediawiki/2016/1/18/T--XMU-China--Design5_and_.png)
In our experiment, the M13 phagemid is constructed by the sRNA cassettes and a phage-derived f1 origin (M13ori) of replication. And the helper plasmid contains M13, coding all the machinery genes required to produce viral particles. When the phagemid and helper plasmid are co-transformed, the sRNA cassettes 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 [3]. The scaffold sequence contains an Hfq-binding motif. Hfq protein helps the combination of mRNA target and the sRNA,and then lead the target mRNA to degrade.
We supposed to design a aac(3)-Id targeted gene. Aac(3)-Id confers E.coli 0157:H7’ resistance to gentamicin(Gm) and sisomicin(Sc) to Salmonella enterica. But for the source limitation, we chose a CAT-resistance E. coli as the targeted bacteria for our experiment to test the efficiency of sRNA silencing . In our experiment, the targeted gene was CAT, which leads the CAT resistance. The sRNA cassettes are competent to knocking down the expression of drug-resistant gene. When joint used with certain antibiotics, the drug-resistant bacteria can be killed.
Switch
![](https://static.igem.org/mediawiki/2016/4/44/T--XMU-China--Design6_and_.png)
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.
![](https://static.igem.org/mediawiki/2016/5/54/T--XMU-China--Design7_and_.png)
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.
![](https://static.igem.org/mediawiki/2016/c/c0/Figure_8_new_version1.1.jpg)
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).
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CONTACT US
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Name: XMU-China School: Xiamen University
Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P. R. China 361005
CONTACT US
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Name: XMU-China School: Xiamen University
Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P. R. China 361005