Difference between revisions of "Team:UESTC-China/Safety"

In order to avoid the occurrence of more serious CIP resistance caused by bacterial leakage, we have adopted a strict engineering kill measure——light-activated suicide switches based on the YF1-Fix blue-sensitive system. It ensures that engineering bacteria can survive only in devices where blue light is present. However, our experiments showed that the activation of the light control system requires a long dark time, which is contrary to our concept of efficient ciprofloxacin degradation, so we decided to adopt a more efficient, faster and less costly UV sterilization system. In fact, our hardware systems are also easier to achieve UV sterilization than light-controlled cracking. Besides, 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 initial design: BBa_K3034012. It consists of the blue-sensitive promoter switch (BBa_K592004, BBa_K592005 and BBa_K2277233) and a lysin gene named Lysep3-D8(BBa_K3034004), a compound protein which can lyse bacteria from the inside and outside of cells[1]. Further, the killer protein can be extracted as an "antibiotic" under suitable conditions. In the latest design, we used an ultraviolet sterilization device instead of a suicide system.

Based on these characteristics, we designed the following experiment:

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 it into

E.coli

BL21(DE3). We added the inducer at the logarithmic phase(OD600=0.5) of

E.coli

to verify the effect of intracelluer cleavage of Lysep3-D8(Fig.1).

Second, we induced bacterial expression(BL21(DE3)) overnight under low temperature conditions (16˚C) to obtain a sufficient amount of protein to verify the extracellular cleavage of Lysep3-D8 to E. coli. The final concentration of IPTG is 0.5mM. We performed two sets of experiments: the control group to which PBS buffer was added and the experimental group to which the supernatant was added(Fig.2.1), the control group to which PBS buffer was added and the experimental group to which the protein of the disrupted cells was added(Fig.2.2).

Third, 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.

Last, we conducted experiments on UV sterilization effects.

Result of the first experiment:

Figure 1 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 Abs600 between the experimental group (+IPTG) and the control group (-IPTG) was larger and larger. That is, the Lysep3-D8 protein inhibited the growth of Escherichia coli (BL21) (inhibition rate was about 30.7%).

Results of the second experiment:

Figure 2.1 and figure 2.2 show that the Lysep3-D8 protein has a significant inhibitory effect on the growth of DH5α extracellularly. Figure 2.1 shows the inhibition rate reaches 91.6% and that of figure 2.2 is 81.6%. We believe that the difference in inhibition rates between the two sets of experimental results is that the multiple proteins contained in the intracellular extract provide additional nutrients to

E.coli

DH5α.

Result of the last experiment：

Result of the last experiment：

From the above experimental results, under the condition of suitable protein expression, the antibacterial effect of Lysep3-D8 protein induced by IPTG strong inducer was not significant compared with the blank control. When we verified the induction intensity of the blue light-controlled promoter, from the experimental results, it did not have a good induction effect compared to the IPTG-inducible promoter.

In order to realize our concept of high efficiency and biosafety, in the realization of hardware biosafety, we finally decided to adopt a low-cost and high-efficiency physical sterilization method, using double-ultraviolet sterilization to prevent engineering bacteria leakage to the maximum extent to ensure the environment. And people's safety. This can promote the further development of hardware, making it more potential to enter the community.

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[2]. 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 futher control our engineered

E.coli

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[3]. 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

E.coli

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.

[1] Shuang Wang, Jingmin Gu, Meng Lv, et al.

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, 2017, Volume 55, Number 5, Page 403
[2] Philippe Marliere.

The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world.

Systems and Synthetic Biology, 2009, Volume 3, Number 1-4, Page 77
[3] Daniel J. Mandell, Marc J. Lajoie, Michael T. Mee, et al.

Biocontainment of genetically modified organisms by synthetic protein design.

Nature, 2015, Volume 518, Pages 55–60

The chassis we used are

E.coli

DH5a,

E.coli

BL21(DE3) ,

E.coli

CICIM B0016,

E.coli

TOP 10 and

E.coli

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.

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 and Hardware)

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;

Final Safety Form