Team:BIT-China/Safety

As we all know, industrial bacterial can secrete several chemicals that has considerable significance for us. However, they are also easily stolen by thieves. What’s more, the pathogenic of bacteria can’t be depressed at this time. The threat of bio-weapon cannot be ignored. Recent years, it is reported that a new form of biology is rising: “Biohackery”. Biohackery allows everyone operation the genome of bacteria in their home or garage with themselves. Without the regulatory of an International Association of Synthetic Biology, we can’t make sure that there is no threat coming from someone’s house or garden, isn’t it?

That’s our work in safety. In fact, we not only followed all the rules in order to make sure that our operation is absolutely safe, but also made some contributions to safety. What we made this year is to provide a new method preventing miss, lose or theft in biology lab.

Q1: What if sequencing does not aim at your lock but the function gene? If so, you lock will lose its function.

A: On sequencing problem, it is obvious that nobody will do nothing but sequence the sample at the first time. Them sample must be nurtured and tested to be the right one, which cannot be realized because our system will prevent that happen.

Q2: If cell division inhibition system comes from engineering bacteria, will it be easy for the bacteria to evolve a method to fight against it? If it is exogenous, will it be harm to the production progress?

A: We plan to use system from bacteria themselves, there is no need to worry about evolution. In fermentation progress bacteria will not be used more than once, there will always be the origin strain in the fermentation tank, the rest will be disposed.

Q3: The difficulty of unlocking the E.co-Lock. (Which aspects of the E.co-Lock have anti-theft and anti-crack function? )

A: First, you must know the E.co-Lock genetic pathway. Second, at the beginning of culturing cells, the medium must be added a certain amount of AHL. In addition, when you add three inducers to try to unlock it, you must know the following: what the inducers are, the order of added inducers, how much each inducer you should add, and how long you should wait until you add next inducer. In a word, the complexity of these aspects will greatly increase the difficulty of unlocking it.

Q4: What’s the advantage of your method comparing with others?

A: Referring to the existing methods, whether to strengthen the regulation or to save the bacteria in safes, the bacteria are all protected from outside, which will waste lots of money and energy. And once the bacteria are lost, they can hardly be got back again, even though they are marked. But our method works inside of the bacteria that can prevent the multiplication of them when they are outside. As a result, the nature of the protection of bacteria can be changed from passiveness by people to activeness by the bacteria themselves, which can not only protect the bacteria effectively but save the unnecessary money in regulation.

Q5: As we all known, Different proteins are degraded in different lengths of time. How can you ensure the time between two added substance can open the gate but not lock it down? Just like, when we add 2, the protein 1 has already been degraded.

A: We can add a tail which can control how long the protein can exist. By this way, we can ensure our project’s function.

Q6: Would the lock losing its function via the gene of the Min system mutated?

A: If the gene of the Min system mutate, other useful genes would change. So I think it makes no sense.

Q7: Did all of you participate in the experiment and master the experimental skills ?

A: Of course! We are a team with the strongest spirit of unity and cooperation. All of us work together to face any possible difficulties both in experiment and life. Thus everyone have a good command of our works.

Q8: The molecules like arac, IPTG and aTC would degrade, resulting in the disappearing of the inducible proteins. Consequently, the E. co lock may close again. How to deal with it?

A: We designed memorial circuits to express the needed inducible proteins continuously, avoiding the open lock turn to close again.

Q9: How we know the proper concentration of arac, IPTG ,aTC and AHL, and the corresponding cellular state?

A: We optimize the concentration repeatedly and use modeling to simulate the cellular state.

Q10: Do you have any problem in ligasing genes？What’s kind of means you apply to ligase genes?

We do have difficulties in assembling biobricks.We have tried many ways to solve this problem including 3A Assembly，Amplified Insert Assembly and gene splicing by overlap extension PCR.

3A Assembly (which stands for three antibiotic assembly) is a method for assembling two part samples and selecting for correct assemblies through antibiotics. 3A assembly uses the restriction sites on the prefix and suffix to assemble part samples. This new composite part will maintain the same prefix and suffix as its "parents" and contain a scar, where the cut and re-ligated restriction sites were stitched together.

Amplified Insert Assembly is a method of "BioBricking" two biological parts (i.e. pieces of DNA) together. This method combines the ease and flexibility of 3A assembly with the reliability of standard assembly. In comparison to 3A assembly, this method can take up to two hours longer Splicing overlap extension by PCR is a convenient method to obtainrecombinant gene.

Q11:Is your lock too simple?

A: Our system is not just three genetic circuits. It was because that the time was limited so we just design three circuits for the IGEM competition. We will keep on discovering new “Transcription factor”and “Chaperone” to extend our system.

Q12: Gene sequencing

A: Although the sequence of the human genome has been completely determined by DNA sequencing, it is not yet fully understood. Future projects and areas of research related to the human genome are seemingly endless.

Q13: How does the AHL generated? What is its specific role is? How do you control the amount of the AHL to make the concentration of the bacteria at an appropriate value?

A: Bacteria can release a class of freely accessible small molecule bacterial cell signaling factors to the surrounding environment - Homoserine Lactone (AHL), AHL can be used as a self-inducing agent in the Qs system. The flora density can be induced by self-induced molecular. About the solution of the AHL, We can optimize the concentration repeatedly and use modeling to simulate the cellular state.

Q14:Since your system is so huge, then will your have room to add something more?

A: Certainly. At first, we put our system into E.coli. by two plasmids. After our system work normally, we will integrate our system into the genome of E.coli. by some experiments. Genome is just like a huge plasmid of E.coli. At that time the scientists will have room to do some other work on E.coli.

Q15: Where you get the date for modeling?

A: We obtain the most data from the experimental results. To some date cannot from the experiments, we adjust these data from the latest literature to make the models more consistent with the experimental results.

Q16：Can we change the promoters to prevent the problem of basal expression?

A: Those promoters could be replaced by other promoters. The aim of our project is to provide a new solution for logic lock in microorganisms; all biobricks in our system are able to be changed if the architecture is intact.