Abstract
When we talk about internal clock, we actually refer to the life cycle of higher animals and plants. People naturally get up when the sun rises and fall asleep as night comes. Thanks to our internal clock, we are able to do appropriate things at appropriate times. This year, we develop a plug-in for microbes, a system that can build up a living clock for microbes and guide them to live regularly as well as to work efficiently. Also, microbes with the system inserted acquire the concept of life span so that some dangerous bacterias will come to death in time before they cause any biohazard. Inverted components which based on recombinase system are introduced in our system. One inverted component is able to calculate a period of time, while several inverted components comprise of one time cycle. When we combine several time cycles together, a sequence with a timer’s function has been successfully formed. Through altering recombinases and their specific sites to different matches, we can get distinctive time cycles. We believe that the system will be of great importance to biological devices with time-related concepts.
Background
Meanings of Timer in Microbes
It is well known that the internal clock is of great significance to human life. The time rhythm let people work and rest regularly, which keeps us healthy and energetic. However, in microbes, especially prokaryotic organism, we could hardly find a strain with rhythm.
Actually, adding rhythm into microbes is a meaningful thing, by which would provide us a brand new technology platform or control system. This year, we hope to construct a rhythm device with time limit. As follows, we will use 3 simple hypothetical cases to illustrate its future application potentials.
a. Industry application of microbial rhythm
When it comes to microbial industry, we often find restrictive reaction space and strict operations necessary, since in the current industrial production, without autonomic consciousness, microorganisms could only behave under stress. So, if we want to express several proteins in turn, this limit determines that we have to add a new inducer once in a while to initiate the expression of a new gene. However, once microbes have autonomic rhythm and the concept of time, any operation with timing demands could be done in a autonomic way. We could make bacteria express proteins in chronological order, or realize certain behaviors by periodic expression, and then, apply to industrial mass production.
b. Advantages of adding microbes rhythm in therapy
Frankly speaking, almost everyone has the experience of not taking medicines on time. In fact, patient compliance has always been a significant problem effecting the therapeutic results. It is common that a patient has to take different pills in different time each day, using the engineered E.coli to secret medicines with rhythm would greatly simplify the medications and improve drug therapy compliance, therefore, ensures the curative effect. What's more, long-termed treatments would become available for any diseases or symptoms associated with time.
c. Meaning of hereditary time limits
By far, many iGEM projects are based on the construction of strains used in medical treatments, but planting living microorganisms into human body has great potential safety hazards. However, if the microbes have time limit devices triggered by inverted modules which would let microbes commit suicide in calculated time, we might be able to use them to treat human. Such microorganisms would die naturally after function, and since the condition of inverted modules is hereditary, the suicide process of the group would not be disturbed by cell fission, which would avoid most of the potential hazards. Time limits are also capable of more sophisticated applications, for example, timed initiation of gene expression. In the future biological industries and biomedical fields, its prospects are almost limitless.
Recombinase System
The design of our project is mainly based on recombinase system, a complete recombinase system comprises of a kind of recombinase and its corresponding recombination targeting sites. Here are the recombinase system's principle.
a. when two recombination target sites (RTS) are in the same direction, the sequences between will be cut out and form a circular DNA under the performances of corresponding recombinase.
b. when two RTS are in reverse direction, the sequences between will be flipped under the performances of corresponding recombinase.
Description
Matching and Testing
The goal of our group is to help provide a systematic solution with optimized synthetic elements to gain a Micro-timer for any length of time. To achieve the goal, we must attain the precise definition and measurement of “time unit”— how long each invertase module exactly represents. Hence, we developed a real-time invertase dynamics testing system to observe the performance of each combination of different elements. The real-time invertase dynamics testing system consists of two different plasmids in E. coli, pInv-gen and pInv-rep respectively. PInv-gen produces invertase-EGFP fusion protein, and pInv-rep produces mcherry signal. Once if the inducer is added into the culture, the green fluorescence will increase at first due to the expression of invertase-EGFP. Then, the red fluorescence is generated because the invertase-EGFP restores the reversed mcherry sequence. The length of interval between green and red indicates the corresponding single timing length of the invertase module. Furthermore, we added a ssra tag to ascertain the degrading of invertase-EGFP. With this system, we successfully tested the activity of some invertase including cre, vika, scre, vcre, dre and dre.
