We aim to protect the natural balance and provides maximal safety. We used "opposite promoter" to design a Bio-Safety system. We want to create ‘firewalls’ between natural and synthetic cells.
We found positive feedback and negative feedback circuits difficult to assemble and need many proteins to assist. Therefore, we wanted to simplify our safety device. Inspired by the calculation pattern of Central Processing Unit of our computer, we constructed the opposite promoter .There is a classic concept included in our device, which is called” complement representation” used in storing values. In this way, we can complete subtraction calculation using addition. In the biological genetic circuit, we can use positive feedback to achieve the effect of negative feedback. We use reverse promoter to suppress forward promoter strength, the absolute value of “opposite promoter” strength decreases to a great extent.
Opposite promoter design and future work
We engineered a regulatory system that will result in expression of growth inhibitors when bacteria escape their intended environment. With this system we reassure that there are as little genetically modified bacteria in the soil and rivers as possible. The designed Kill-switch system consists of one plasmids with two promoters. Reverse promoter which is opposite to forward promoter is a IPTG inducible promoter. Forward promoter is a constitutive promoter. We nicknamed it as “device” which is shown onfig1.The advantage of our device is that it has short response time because it is affected in transcriptional level. Secondly, we utilize “opposite promoter” to suppress the expression of toxin protein greatly. When the bacteria escape or plasmid drift, bacteria will lead to death without the induction of IPTG. The higher the reverse promoter strength, the better outcomes we will get.
Figure 1│Pathway design of opposite promoter.
Promoter design results at first stage
We worked on the construction of "opposite promoter" with Anderson promoter “BBa_J23100” and ”BBa_J23106” .We were able to assemble “opposite promoter” with RFP to characterize its functionality for an improved Kill-switch. The visual selection was demonstrated on the Figure 2.
Figure 2│Promoter design results at first stage.
Figure 3│ New constructed promoters upstream RFP, expressed in E. coli. DH5. There are three samples by precipitating bacteria through centrifuge. A：DH5αwithout plasmid B：DH5αwith opposite promoter C：DH5αwith J23100 single promoter
 Wright O, Stan GB, Ellis T. Building-in biosafety for synthetic biology. Microbiology 2013;159:1221-35.