Before we began our project, we first analyzed the potential risks we might encounter. And we finally came to the conclusion that our risks mainly came from three parts: 1) the organism we use; 2) the lab activities; 3) risks during implementation.
This year, we choose a quite normal organism in the environment, Bacillus subtilis, as our chassis. According to the check-in form, Bacillus subtilis can be classified as Risk Level 1 organisms, which means that it has low risks affecting our health. Therefore, we assess that the overall risks are low. However, in order to reduce horizontal gene transfer and avoid leaking out, we still find it necessary to design a kill switch for our engineered bacteria.
Of course, our lab activities can exert a certain degree of security risk, which we must try to avoid.
Besides, if our project can develop into real use, one thing can't be overlooked is the harm our product may cause to the stone relics.
To ensure biosafety, it is necessary to prevent our modified bacteria from posing a threat to the surrounding environment. In our design, we use the toxin-antitoxin system documented in the iGEM Parts (BBa_K2292006) for our design. Since the system is mainly expressed in E. coli, we use the sucrose-inducible promoter from Bacillus subtilis to adapt our chassis and modify it.
The toxin-antitoxin system depends on a pair of proteins, Maz E and Maz F. MazF is a long-lived toxin that can nonspecifically cleave intracellular RNA and cause cell death. MazE is an unstable antitoxin that antagonizes MazF and can be degraded by ATP dependent ClpPa serine protease in cells. Due to the characteristics of MazE/F, only the continuous expression of Maz E can prevent cell death.
In order to achieve the suicidal function,we introduce a sucrose induced promoter PsacB. It’s the third commonly used promoter system in Bacillus subtilis. The expression activity of this Bacillus subtilis endogenous promoter in the presence of sucrose is 100 times higher than that in the absence of sucrose, which is used to regulate the expression of downstream MazE/F module.With the increase of culture time, the concentration of sucrose in the medium will decrease, the activity of PsacB will decrease, and MazE cannot be continuously expressed. Based on its instability, its intracellular concentration will decline faster than MazF, resulting in MazF playing its toxic role and eventually leading to cell death.
PsacB promoter can be divided into two parts with more detailed functions in theory: the narrow sense promoter which can only recruit the transcription complex, and the leader RNA sequence which plays a regulatory role.
During the experiment, we found that PsacB has 3 disadvantages: 1)The transcription of the PsacB promoter is not strictly regulated by sucrose, and can be transcribed without an inducer. 2)The expression of PsacB is not high. 3)Under different conditions, PsacB’s expression is not very stable.
To improve this part, we replace the narrow sense promoter sequence of PsacB with the strong promoter Pveg and design a tandem repeat of the Leader RNA sequence, with an added spoVG RBS at the end.
Fig 1 The gene circuit design and improvement of safety module
In order to reinforce the management of the labs, the official of our university delivered management regulations. All of our activities are instructed under the official guidance. Besides, our lab also special regulations on management of instruments and equipment, which we must obey during experiments.
To gain access to our lab, we must apply on the lab website and take a test. The test included basic experiment skills, lab cleaning, experimental waste disposal, ethics, chemical reagents storage, lab rules, equipment safety, and emergency measures. Only those who pass the test are allowed to carry out experiments in our lab.
Associate Professor Yang Fan is the instructor of our lab. She has been in charge of our lab for 8 years. She is familiar with most molecule cloning experiments and can offer suggestions at any time. So our experiment specification is under the control of the instructor in order to avoid any possible danger. Before we began our business in the lab, we also had a 'safety education class' delivered by her to train us the specific rules to follow in the lab and how to deal with the possible emergency. When we are carrying out our experiments, our instructor will supervise our experimental operations,decreasing the posibility of unnecessary danger and damages.
Fig 2 A glimpse of our experiment bench
Fig 3 The clean bench for sterile operation
As the stone relics are of great value, our project must avoid causing significant damage to them when we try to repair the tiny cracks.
There are several challenges we may meet when the project is put into practice.
First of all, the medium we should use is of high concentration of sucrose and calcium ion. In consideration of the fragility of the historical relics, we must carefully design the substance involved and their concentration
When we inject our medium into the tiny cracks, our engineered bacteria may disturb the original biotic environment and produce undesirable metabolites, which may corrode the stone relics. Therefore, we must further regulate the growth of our organism before use, reducing the secretion of metabolites
To avoid secondary damage to the relics, our devices for restoration must be specially designed, as well as to achieve better restoration effect.
