Basic Part
·PhiX174E from phage PhiX174E
Figure 1. Lysis Effect of PhiX174E Gene on Bacteria
Locus: CAA84691, 91 aa, linear PHG 18-APR-2005
https://www.sciencedirect.com/science/article/abs/pii/037811199400839K
The lysis gene E of phage PhiX174 (PhiX174E) is a 91-amino acid membrane protein, which is lethal for E. coli cells by inhibit cell wall synthesis. In recent years, people use PhiX174E to design suicide switch and set up many genetic circuits. In our design, in order to serve the purpose that our engineered bacteria could release those degrading enzymes in a regular cycle, we introduced a synchronized lysis circuit (SLC), in which PhiX174E is contained. And considering biosafety, we designed a heat-inducible suicide switch by PhiX174E in our engineered bacteria.
Reference:
[1]Bernhard Henrich, Brigitta Schmidtberger,. Positive-selection vector with enhanced lytic potential based on a variant of pX174 phage gene E, ScienceDirect, Volume 154, Issue 1, 27 February 1995, Pages 51-54
·Cytochrome P450 (CYP) and Ferredoxin Reductase (FR) from Rhodococcus rhodochrous.
Figure 2. The Degrading Process of Guaiacol
CYP gene MK007067, FR gene MK007068
https://amb-express.springeropen.com/articles/10.1186/s13568-019-0759-8#Tab1
Is known to proceed initially via demethylation to catechol, and this reaction is catalyzed by cytochrome P450 monooxygenases. These enzymes typically require a set of redox partner proteins, whose number and identities were not described until very recently in the case of guaiacol. In a 2019 AMB Express work, the authors identified FR is a great partner and combined expression of CYP and FR enables P. putida to metabolize guaiacol. In our design, CYP and FR are used to degrade guaiacol.
Reference:
[1]Javier García-Hidalgo, Krithika Ravi, Lise-Lotte Kuré, Gunnar Lidén & Marie Gorwa-Grauslund, Identification of the two-component guaiacol demethylase system from Rhodococcus rhodochrous and expression in Pseudomonas putida EM42 for guaiacol assimilation, AMB Express, Volume 9, Article number: 34 (2019), March 11 2019.
·Monoamine Oxidase (MAOA) from Escherichia coli str. K-12 substr. W3110 & Aldose reductase (ALDR) from Escherichia coli str. K-12 substr. MG1655
Figure 3. The Degrading Process of Serotonin
MAOA gene BAA04900, ALDR gene b3011
https://pubmed.ncbi.nlm.nih.gov/19609521/
https://www.sciencedirect.com/science/article/abs/pii/0922338X94902410
The structural gene for monoamine oxidase (MAO) was located at 30.9 min on the Escherichia coli chromosome. The mature enzyme (M.W. 81,295) can catalyze serotonin (5-hydroxytryptamine, 5-HT) to 5-HIAL. The ALDR gene is originally used as an agent to produce biofuels. In the above figure, ALDR can catalyze 5-HIAL to 5-HTOL. In our design, ALDR and MAO are used to degrade serotonin in the intestine of locust.
Reference:
[1]Shota Atsumi, Tung-Yun Wu, Eva-Maria Eckl, Sarah D Hawkins, Thomas Buelter, James C Liao,. Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes,. National Library of Medicine, Volume 85, Issue 3, 2010 Jan.
[2]Hiroyuki Azakami, MitsuoYamashita, Jung-HyeobRoh, Hideyuki Suzuki, Hidehiko Kumagai, Yoshikatsu Murooka,. Nucleotide sequence of the gene for monoamine oxidase (maoA) from Escherichia coli, ScienceDirect, Volume 77, Issue 3, 1994, Pages 315-319.
Composite Part
·Abstract
This year, we used a total of 6 composite part, including a heat-responsive RNA-based fluorescent measurement device, a heat-responsive self-lysis circuit, a quorum sensing synchronized self-lysis device, a quorum sensing fluorescent reporting device, an guaiacol degradation device and serotonin degradation device.
Figure 4. gene circuit of heat-responsive suicide switch
BBa_K2541001:
Heat Responsive RNA-based Fluorescent Reporting Device
BBa_K3594001:
Heat Responsive Self-Lysis Circuit
The heat inducible suicide switch is constructed on the pSB1C3 vector by T4 ligation. The suicide switch (BBa_K3594001) is composed of thermal sensor (BBa_J2541001) and a self-lysis gene PhiX174E. At the same time, to demonstrate if the RNA-based thermal sensor works well, we design another plasmid with sfGFP as a reporter (BBa_K3594002). Originally, we chose BBa_J23104 as our promotor.
·Quorum Sensing Device
Figure 5. Genetic Circuit of Quorum Sensing Device
BBa_K3594002
:Quorum Sensing Synchronized Self-Lysis Circuit (LuxR_Phix174E)
BBa_K3594003
:Quorum Sensing Fluorescent Reporting Device (LuxR_sfGFP)
They are two devices for quorum sensing of the bacteria. They are constructed on the pSB1C3 vector by Gibson Assembly, and they are composed of Plux promotor, LuxR sequence, Phix174E sequence or sfGFP sequence. The promotor of these two circuits are controlled by LuxR-AHL signal molecules, which are synthesized by the bacteria themselves.
·Guaiacol and Serotonin Degrading Device
Figure 6. Gene Circuit of Guaiacol and Serotonin Degrading Device
BBa_K3594004
:Guaiacol Degrading Device (pTac_CYP_FR)
BBa_K3594008
:Serotonin Degrading Device (pTac_MAO_ALDR)
They are two devices for guaiacol and serotonin degradation, which respectively contains CYP&FR sequence and MAOA&ALDR gene sequence, T7 promotor and RBS, and they are constructed on the pSB4K5 vector by T4 Ligation. The promotor of these two circuits are controlled by LuxR-AHL signal molecules, which are synthesized by the bacteria themselves.