In order to avoid the occurrence of more serious CIP resistance caused by bacterial leakage, we have adopted a strict engineered killing measure - double safety sterilization system. In the first security mechanism, we adopted a photo-activated suicide switch based on the YF1-Fix blue-sensitive system to ensure that E.coli can only survive under blue light conditions and be killed in the dark ; in the second security mechanism, we used the UV sterilization technology to kill all harmful bacteria and ensure that no other super bacteria are produced. With such a security system, biosafety can be guaranteed to the greatest extent. Further, to avoid, prevent and minimize the information coded by the antibiotic resistance being passed onto the environment that known as horizontal gene transfer, we can use semantic containment methods to achieve a more safe and effective biological containment.
This is our first security mechanism: BBa_K3034012
. It consists of the blue-sensitive promoter switch (BBa_K592004
) and a lysin gene named Lysep3-D8(BBa_K3034004
), a compound protein which can lyse bacteria from the inside and outside of cells , and theoretically has a stronger effect of lysing bacteria.
First, amplifying Lysep3-D8 gene from a plasmid containing BBa_K3034012
, we inserted it into an expression vector induced by IPTG(final concentration is 0.5mM) and transformed the recombinant plasmid into
BL21(DE3). We added the inducer at the logarithmic phase(OD600=0.5) of
to verify the effect of cleavage of Lysep3-D8 (Fig. 1).
Second, we inserted red fluorescent protein (TagRFP) after the light-sensitive promoter system to characterize the function of the light control system. The control group was under continuous illumination at 470 nm and the experimental group was in absolute dark conditions(all other conditions are exactly the same). After 24 h, comparing the experimental group with the control group, we found that the experimental group did not show the expected red color visible to the naked eye. In the future, we are going to optimize the light-controlled promoter with methods such as site-specific mutation.
Result of the first experiment:
Fig. 1. Characterization of intracellular cleavage effect of Lysep3-D8 protein(37˚C) in E.coli BL21(DE3). The experimental data comes from two sets of biological replicates, repeated three times in each group.
Our experimental data shows that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in OD600 between the experimental group (IPTG+) and the control group (IPTG-) was obvious (Fig. 1). That is, the Lysep3-D8 protein obviously inhibited the growth of
(BL21) (inhibition rate was about 30.7%).
Finally, combined with the second safety mechanism-UV sterilization mechanism, we have minimized the risk of leakage of engineered bacteria and production of harmful bacteria. We provide meaningful advice for hardware in biosafety design.
Horizontal Gene Transfer(HGT)
To minimize the risks associated with bacterial release, we'd better promise the biosafety from three mechanisms, mutagenic drift, environmental supplementation and horizontal gene transfer (HGT). At present, there are three main solutions to the problem of horizontal gene transfer of genetically modified organisms: nutritional containment, heteronuclear acid and semantic containment . The control of nutrient containment may be attenuated or invalidated by the supplementation of natural compounds. The heterologous nucleic acid method is difficult to artificially insert artificially synthesized bases in biological nucleic acids, so we will use semantic containment methods to further control our engineered
in the future.
Its main principle is to transform the genetic sequence of the organism so that the UAG triplet codon is no longer a stop codon, but can encode an amino acid. In order to ensure a high probability of low leakage. UAG is encoded into artificially modified amino acids, rather than natural amino acids, are effective . Studies have shown that this method is achievable and very powerful, so we believe that the same method can be used to prevent the leakage of resistance genes in our experiments, and the essential genes of
such as the enzyme III subunit δ (holB), methionyl-tRNA synthetase (metG), phosphoglycerate kinase (pgk), etc. can be modified to encode the codon UAG into NSAA L-4, 4'-biphenylalanine (bipA), which has a different size and geometry than any standard amino acid, as well as hydrophobic chemicals that are expected to be compatible with the protein core.
Wang, G., Lu, X., Zhu, Y., et al. (2018). A light-controlled cell lysis system in bacteria. Journal of industrial microbiology & biotechnology, 45(6), 429-432.
Wang, S., Gu, J., Lv, M. et al. (2017). The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region. Journal of Microbiology, 55: 403.
Marliere P. (2009). The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world. Systems and Synthetic Biology, 3(1-4):77–84.
Daniel J. Mandell, Marc J. Lajoie, Michael T. Mee, et al. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518: 55-60.
The chassis we used are
TOP 10 and
MC1061, which all belong to RISK GROUP 1, means they are low risk for human being and environment.
Our parts are all taken from the RISK GROUP 1 and RISK GROUP 2 organisms. The RISK GROUP 1 organisms are not safe to cause diseases in healthy adults. Although the RISK GROUP 2 organisms may cause diseases in humans, the parts we extracted were all synthesized from the company, without any risk of pathogens.
Expected Protection Mechanism
The greater risk of our project is that genetically modified bacteria that carry antibiotic resistance can escape into the wild under incorrect operation. Although the transgenic bacteria we used don't cause disease themselves, they can still transfer plasmids that carry antibiotic resistance to those pathogens. Therefore, UV disinfection lamps are installed in our hardware devices for biosafety. (see more at Biosafety
To avoid the pollution of environment, we gather all cultivate medium with bacteria and sterilize them before discarding. For some reagents with health risk, such as TEMED, we use them carefully with the correct position and keep another separate container for hazardous waste. In order to keep our laboratory safe and clean, we carry out regular cleaning, sterilization, and disinfection every day. Besides, we also check out the electrical installment and instrument to ensure safe experiment and environment. Ultra-clean working table is treated with ultraviolet sterilization before use.
We had a lesson about lab safety provided by the university biosafety office in our freshman year. In this lesson, freshman can learn the correct experimental operation and much knowledge about chemical drugs and reagents. And the students who pass the final safety test will accept a diploma, with which one may enter the lab and start experiment.
Our experimental operation strictly in accordance with the guidelines:
Laboratory coveralls, gowns or uniforms must be worn at all times for work in the laboratory;
When some volatile toxic reagents are necessary, we will operate in the fume hood;
All reagents have designated position and must returned after experiment;
A variety of drugs and reagents must be signed a clean label including the name, concentration, specification, etc.;
Daily decontamination of all work surfaces when work is complete;
Prohibition of food, drink and smoking materials in lab setting;
Pipetting by mouth of any material is forbidden. You must always use the teats, syringes, and pipette-fillers provided;
Contaminated glassware, plastic ware, microscope slides and discarded Petri dishes etc., must be placed in the receptacles indicated by the lecturer in charge;
IGEM Safety Form