Figure 1. The mind map of biosafety design
In order to protect natural ecosystems also our patients for microbial therapies, and address public concern, biosafety is a critical concern in the development of our Ark.micro. We concerned biocontainment, avoiding unintended proliferation into the environment, also biosafety, which we mainly concerned avoiding its escaping from the specific lesion to other healthy parts of the body. Current strategies mainly include integrating toxin/antitoxin ‘kill switches’ and establishing auxotrophies for essential compounds. However, either of them has its own obstacles. This year, we provided our own solutions and improvements to address the risks that may cause our ark’s sinking. By combining our high-performance cold-inducible on-switch with the toxin system, we optimized the ‘kill switch’ from its foundation, improving the response speed also the efficiency. By developing a ‘synthetic auxotrophy’ for non-canonical amino acids, our therapeutic bacteria are robust against environmental supplementation. Additionally, considering the dependence on exogenous source, we innovatively combined our heat-inducible on-switch with ncAA system, exploring greater potential for our ark to sail to the real world.
Cold-inducible On-switch + Toxin
One way to address the biosafety issue is using toxin-antitoxin pairs. Toxin is a kind of protein which is the output of the genetic circuit. Under certain condition such as temperature, toxin begins to be expressed by the engineered bacteria and eventually kill those bacteria.
In our engineering bacteria, we used Doc, a toxin interferes with basic metabolism at the level of translation, to associate with our cold-inducible on-switch, so that under low temperature, the switch is turned on to express Doc protein.
Figure 2. The design of establishing our cold-inducible kill switch.
To explore the possibility of escape for evaluating the performance of our kill switch, we measured the growth of the bacteria with DOC gene in LB solid medium at different temperatures and dilutions. The results showed that the strain cultured at 25℃ grew much worse compared with the strain grown at 37℃, and the 20h average solid escape frequency is 2.318×10^(-2) , the lowest escape rate can be limited to 10^(-3) level.
Figure 3. The escape rate of bacteria on solid LB plate after 20h. The escape rate is calculated using the formula 𝒑𝒆𝒓 𝑬𝒔𝒄𝒂𝒑𝒆 𝑭𝒓𝒆𝒒𝒖𝒆𝒏𝒄𝒚 =(𝑪𝒐𝒍𝒐𝒏𝒊𝒆𝒔 𝒐𝒏 𝒏𝒐𝒏𝒑𝒆𝒓𝒎𝒊𝒔𝒔𝒊𝒗𝒆 𝒑𝒍𝒂𝒕𝒆 × 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏)/(𝑪𝒐𝒍𝒐𝒏𝒊𝒆𝒔 𝒐𝒏 𝒑𝒆𝒓𝒎𝒊𝒔𝒔𝒊𝒗𝒆 𝒑𝒍𝒂𝒕𝒆 × 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏) , the escape rate of 3 groups are (1×10^7)/(2×10^8 )=5×10^(-2) , (11×10^7)/(6×10^10 )=1.83×10^(-2) and (2×10^7)/(16×10^9 )=1.25×10^(-3) respectively(from above to below). And the 𝑬𝒔𝒄𝒂𝒑𝒆 𝑭𝒓𝒆𝒒𝒖𝒆𝒏𝒄𝒚 = 𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝑬𝒔𝒄𝒂𝒑𝒆 𝑭𝒓𝒆𝒒𝒖𝒆𝒏𝒄𝒚 ± 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝒅𝒆𝒗𝒊𝒂𝒕𝒊𝒐𝒏 =2.318 ×10^(−2) ± 0.122.
Moreover, we measured the growth curve of the strains with DOC gene at different temperatures to characterize their growth in liquid medium, and took the strains without doc gene as the control to verify the effectiveness of the toxin system. The results showed that the growth of the two strains was almost the same at 37℃, indicating relatively low leakage of the system and low while the growth of the strains with doc gene was worse at 25℃, indicating that the system with DOC as toxin was effective.
Figure 4. The growth curve of bacteria on liquid LB. Incubated in 4 mL volume in 24-deep-well plate.
Based on the design of our cold-inducible switch and integrating toxin Doc, we developed a ‘kill switch’ which sensitively and accurately responds to the natural signal of human body and the environment – temperature. We successfully limit the escape rate to 10^(-2) to 10^(-3). Considering the improvement of the robustness and performance of the whole system, also to ensure the ability of our ark to adapt to various situation with as little risk for both human and nature as possible, we design another strategy as follow.
Currently, another biocontainment strategy is establishing auxotrophies for essential compounds, however, metabolic auxotrophies can be circumvented by scavenging essential metabolites from nearby decayed cells or cross-feeding from established ecological niches. By establishing ‘synthetic auxotrophy’ for a non-canonical amino acid, we introduced a robust and effective strategy against environmental supplementation, which is considered as the major problem of current auxotrophy strategy.
We assigned the TAG stop codon to incorporate a noncanonical amino acid and tried to redesign an essential gene to develop corresponding dependent bacteria.
Figure 5. The general design of establishing our ‘synthetic auxotrophy’. pEVOL plasmid is a vector with aaRS and tRNACUA, it can help incorporate the ncAA into proteins in Escherichia coli with increased yields and stability. We assigned the TAG stop codon into an essential gene so that only when the bacteria are provided with the ncAA, the essential enzyme can be synthesized and support the survival of the engineered bacteria.
Getting a high-performance aminoacyl-tRNA synthetase (aaRS) system from our PI’s laboratory, we chose 3,5-dichloro-L-tyrosine (Cl2Y) as the noncanonical amino acid. First, we proved the concept of our design using GFP as the target gene. We constructed mutant GFP with 9TAG and 17TAG as the potential incorporating site. When additionally providing Cl2Y, we could see a prominent increase(~100 folds) of the fluorescence, indicating the high efficiency of incorporation.
Figure 6. When provided Cl2Y, the fluorescence of GFP can be increased almost 100 folds, indicated the high efficiency of incorporation.
MS result further proved that Cl2Y was incorporated into the target site with high selectivity and efficiency.
Figure 7. Mass spectrum result of 9Cl2Y GFP. 27824 indicates the molecular weight of interested protein.
Figure 8. Mass spectrum result of 17Cl2Y GFP. 27794 indicates the molecular weight of interested protein.
For the essential gene, we first constructed a ΔBioB strain by knocking out the BioB gene of the bacteria, the key enzyme in the last step of biotin synthesis. By incorporating TAG codon into 288 site of the enzyme, we successfully engineered a non-canonical amino acid dependent bacteria strain.
Figure 9. The circuit for establishing our ‘synthetic auxotrophy’.
We assumed that even entered the non-permissive environment, the escape rate is expected to be limited in a controlled and ideal rate. However, due to the time limitation, we have to keep this part in our future work.
Heat-inducible On-switch + ncAA
For our therapeutic bacteria, exogenous source of non-canonical amino acid may constrain its function. So, we designed a thermo-sensitive non-canonical amino acid producing part, by combined our heat-inducible on-switch with the synthetase of our targeting non-canonical amino acid, we can strictly support the growth of our bacteria only inside the human body.
We considered to use tyrosine hydroxylase to synthesize L-dopa as the non-canonical amino acid. When TCI induces the endogenous supplement of L-dopa under 37℃ circumstance, proper translation, folding and function of biotin synthase BioB will be achieved, our ark can navigate safely to arrive its destination.
Figure 10. The design of heat-inducible on-switch combined with ncAA system for our ‘synthetic auxotrophy’, providing a solution for the current obstacles of dependence on exogenous source of non-canonical amino acid.
We successfully synthesized L-dopa in the proper situation, this part of result can be seen in PROJECT-PARKINSON'S DISEASE. With a developed DHPRS and tRNA¬CUA system, we can incorporate L-dopa into the intended site, as you can see in the mass spectrum below.
Figure 11. Mass spectrum result of 17L-Dopa GFP. 27726 indicates the molecular weight of interested protein.
However, due to the limitation of the system itself, the selectivity and efficiency of the L-dopa incorporation were not as good as our expectation. We could predominantly reduce the background by adding TAG codons, but it was a pity that we did not have enough time to replace Cl2Y as L-dopa to complete the missing link, we are confident that it can be complete in our future work based on the performance improvements of the DHPRS.
Figure 12. Relative Fluorescence of 17+3TAG GFP with/without L-Dopa. The background was predominant controlled by assigning another TAG stop codon into the interested protein.
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