Team:Tsinghua/Design
Design
NOS cloning
Nitric oxide (NO) is a key molecule that induces biofilm degradation. Nitric oxide is produced by Bacterial nitric-oxide synthases (bNOSs), which converts arginine into NO. In bacteria, since there is no corresponding reductive domain, additional reductive substances are needed for reduction. ALA is a heme precursor that associate with the corresponding reducing substrate.
In our experiment, we first cloned NOS gene in Bacillus subtilis by PCR and attached it to Pet28-a for determination using double restriction enzyme and DNA ligase. The Pet28-a has a T7 promoter, which is a strong promoter. It also has a kanamycin resistance to help us to screen the transformed cell. It also has a lacI gene and lac operator, which make us to use IPTG to induce NOS expression. Meanwhile, there is a his tag in the vector, which can be used for the further detection. More details in Experiments and Results.
C4-HSL quorum sensing
As our expectation, the engineered bacteria could sense the C4-HSL signal derived from P . aeruginosa. Thus, a C4-HSL report system should be considered and established. Here we characterized the composite part BBa_K1893003 as the report system. Since this part was not distributed, it was synthesized in BGI QINGLAN BIOTECH according to the sequence offered on the website and then integrated into plasmid pSB1C3. (The backbone of pSB1C3 is derived from distributed part BBa_R0071) The work condition of the composite plasmid was tested in different C4-HSL strains and different C4-HSL concentration.
Note: the Rhl and pRhl are WT and not improved with relatively lower expression level, compared with the part improved by iGEM 2014 Tokyo_Tech.
More details in Experiments and Results.
Assemble of C4-HSL-NOS system
For our last-step assembly, it is expected that the engineered bacteria could sense the C4-HSL signal and then trigger the expression of NOS gene. This part was synthesized via HiFi assembly and transformed to DH5ɑ and BL-21. Due to an unexpected point mutation(Lys→Glu) in the ORF of NOS gene, it took us some time to fix this mutation. The sequence result showed the success of our engineering, but due to the limited time the characterization of this element is still needed to be finished.
More details in Experiments.
Assemble of NO reporter system
The NO-sensing system is proposed to monitor the progress of the NO promoted biofilm dispersion and verify the production of NO. It’s made of the hmp promotor phmp, which has been testified that can be activated by NO or other nitrogen oxides and the reporter gene EGFP. The phmp sequence was cloned from E.coli genome and the EGFP was cloned from pEGFP plasmid. Here we tried 2 hmp promoter with different length, according to the database and previous studies, and detected its activity. However this these two promoter still not work in both DH5ɑ and BL-21, indicating that some vital factors was missed.
More details in Experiments.
Suicide Switch
Overview
For biosafety, we don't want engineered bacteria to escape from the tube. Thus, we designed a light-activated suicide switch. When the engineered bacteria escaped out of the tube, they would be exposed to natural light and then die. We used the eLightOn system and the miniColicin(a kind of nuclease) to construct a kill switch, which could trigger the engineered bacteria to commit suicide when exposed to blue light with a peak wavelength of 460nm. We have designed the components of the kill switch, and the construction and testing will be followed up in future.
Design the kill switch controlled by eLightOn system
We used eLightOn system as the light-regulated modules.[1]The eLightOn system is a single-component systems consisting of single transcription factors. In the eLightOn system, the synthetic light-switchable repressor LexRO is based on a novel LOV light sensor domain RsLOV. In darkness, LexRO dimerizes and binds to its LexA408 cognate operator sequence to repress promoter activity. Upon light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates gene expression. LexA408 cognate operator sequence was incorporated into a constitutive promoters, ColE.[1]We used the eLightOn system to control the expression of miniColicin, the Colicin E2 DNase domain which is toxic to coliform bacteria. MiniColicin is a nicking endonuclease.(shown as Figure A and Figure B)
Figure A. Schematic diagram of the kill switch controlled by eLightOn system.
Figure B. Elements of the kill switch controlled by eLightOn system
Reference:
[1]Xie Li, Changcheng Zhang, Xiaopei Xu, Jun Miao, Jing Yao, Renmei Liu, Yuzheng Zhao, Xianjun Chen, Yi Yang, A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells, Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, Page e33, https://doi.org/10.1093/nar/gkaa044
[2]E. Hernández-Urzúa · D. S. Zamorano-Sánchez ·J. Ponce-Coria · J. Membrillo-Hernández, S. Grogan · R. K. Poole, E. Morett. Multiple regulators of the Flavohaemoglobin (hmp) gene of Salmonella enterica serovar Typhimurium include RamA, a transcriptional regulator conferring the multidrug resistance phenotype. Arch Microbiol (2007) 187:67–77
[3]Pulin Liu Q H W C . Liu P, Huang Q, Chen W. Heterologous expression of bacterial nitric oxide synthase gene: a potential biological method to control biofilm development in the environment[J]. Canadian journal of microbiology, 2012, 58(3): 336-344.[J]. Canadian Journal of Microbiology, 2012, 58(3):336.
[4]Gusarov I, Starodubtseva M, Wang Z Q, et al. Bacterial nitric-oxide synthases operate without a dedicated redox partner[J]. Journal of Biological Chemistry, 2008, 283(19): 13140-13147.
[5]http://parts.igem.org/Part:BBa_K1976048
[6]http://parts.igem.org/Part:BBa_K1893003
