Matching and Testing

Introduction of purpose

The basic idea of Micro-time system is to separate a long-period timing into small invertase device modules, and through appropriate combination of them, we can obtain a wide range of aimed time length for users to choose. However, for both E. coli and yeast, a successful timer must be based on precise definition and measurement of “time unit” – how long each invertase module exactly represents. Hence, the major consideration of our testing group is to measure the time unit for different invertase modules, and provide a systematic solution with optimized synthetic elements to gain a Micro-timer for any length of time (see Fig-T1).

This mission require us to utilize a method to quantitatively understand the in vivo enzymatic dynamics of each invertase, and the system we conduct such experiment must be reconstruction-friendly, since we have to test a variety of elements (e.g. promoter and ssra) in similar pattern to most accurately obtain data. Hence, we developed a real-time invertase dynamics testing system to observe the performance of each combination of different elements.

System construction

The real-time invertase dynamics testing system contains two different plasmids in E. coli (see Fig-T2). The first one is an invertase generation vector, namely pInv-gen, that produces invertase-EGFP fusion protein through induction. The second one is called pInv-rep, a reporter vector that produce mcherry signal to indicate the inversion successfully happens. The invertase-EGFP on pInv-gen is controlled by an inducible promoter (T7-LacO promoter or Pbad). The target sequence (RTS) of corresponding invertase locates in the pInv-rep, surrounding a mcherry gene which is yet upside-down and transcribed by a constructive promoter (e.g. BBa_J23101). This mcherry-coding sequence can be inverted and restored to 5’ – 3’ direction at the existence of Cre-GFP, rendering red signal. Additionally, an ssra tag that intensifies the protein degradation may be fused to the C-terminus of invertase-EGFP and mcherry to be in tune with our final device that aims to clean up the redundant invertase not participating in a second round inversion.

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 Cre-EGFP restores the reversed mcherry sequence (see Fig-T-3). The length of interval between green and red indicates the corresponding single timing length of the invertase module. In our study, the variants to render different time length are invertase itself, promoter, and the degrading rate by ssra. Specifically, the activity level of invertase directly determines the time need to invert most of pInv-reps, and the promoter decides the rate of generation of invertase, which also contribute sigfificant to the speed of module. The ssra-tag, on the contrary, reduces the speed of inversion while effectively inhibiting the leakage expression when inducer is not in the culture.

Achievement

We uses this system to measure totally 21 pairs of different combination of pInv-gens and pInv-reps. There are 6 different invertases we have tested using the Real-time system. While Cre and Flp are most commonly used recombinases in Biobrick plates, we newly contributed 4 brand-new invertases: Dre, Vcre, Scre, and Vika, all of which are Cre-family recombinases with different and non-intervolving RTS. We successfully proved that all these invertase work pretty good in our system, which you can see in RESULTS. All of the data we gather are analyzed by modeling group to render its corresponding time length. This work could guide other groups for their final design.

Timer design plug-in

Additionally, we prepared a website plug-in to for potential users to design their own timer with specific length of counting time, a project in cooperation with SYSU-software. According to the data gathered in this research and other promoter intensity given by iGEM official page, we can anticipate the overall timing length of any given combination of various invertase module. Vice versus, if a user could provide his/her target time, we can automatically generate one or more optimized design of Micro-time to precisely match the demand.

Bacteria Timer

Introduction

A report from Science[1]（在本版面最后添加参考文献）, by which we were inspired, tries to explain that synthetic gene networks can be constructed to emulate a cellular counter that would enable complex synthetic programming and a variety of biotechnology applications.

One of the figures (fig. 1) from this article indicates how genes can work in a counting system by reversal of recombinases. Two recombinases in the circuit, Flpe and Cre, in conjunction with their specific recognition site, FRT and LoxP, accomplishes the whole flipping process. Convenient to distinguish, we'll call it circuit 1.

Based on Circuit 1, we designed what we call circuit 2 (fig. 2) , which is, to our perspective, more functional and less induced, by principally altering the location of genes.

The comparison between them indicates 2 advantages of circuit 2 over circuit 1 (fig. 3) .

First, it continues transcripting and flipping in a circulation once induced. It happens theoratically because it may be bothered by objective resistence, but it provides us with a possibility to time gene reaction and control certain protein expression in a time scale.

Second, circuit 2 can transfer certain DNA sequences unit after unit. Imagine if there is a target gene between the first frt and loxP in the initial phase, it would pass on and on in the continuous units after flipping.

Construction

Due to time limits, we focused on constructing circuit 2, our original design.

We added florescent protein eCFP（标上砖号） and mCherry（标上砖号） right in the downstream of the ssrA-tag of recombinase flpe and cre, respectively. And we add a final GFP（标上砖号） as a reporter. pSB1C3 was used as vector for cloning and we tried to transfer the entire circuit to pSB3K3 in order to test its viability (fig. 4) .

In terms of ligation efficiency, we resembles small fragments (promoters, FRTs and LoxPs) and deliver them to IDT for complete synthesis. Then we ligate long fragments in between according to our design (fig. 5) . Worth of attention, we created a new "2A" assembly by using DNA clean up and arranging gene segments with different resistances (fig. 6).

Testing

Experimental proof must be accomplished after construction.

2 objectives for circuit 2 must be achieved during testing. One is that we must prove it actually has capability to reverse. The other one is that we must test its efficiency and explore how close it is to an ideal biological device that can really calculate time.

For the first goal, we thought that it could be solved by digestion (fig. 7) . When sequences flip, certain restricted enzyme cutting sites won’t change. What have changed are their locations. Gene length can be altered when sequences reverse, which can be visualized in an elecrophoretic way, as shown in fig. 7.

For the second goal, we used qPCR to verify feasibility of the circuit. We design primers shown in the picture (fig. 8) . We can tell from amplication curves (fig. 9) whether it reverses and calculates time when phases have altered.

Yeast Timer

(Our project, micro-timer, is to construct a counter on DNA that can imprint time on microbes.)

Micro timer 2.0 (Eu-timer) is constructed by DNA-based counting motifs that are inserted into different sites of chromosomes, creating a relatively large-scale system with more motifs than that in Micro timer 1.0.

The Eu-timer uses recombinases from Ser family such as Bxb1, which typically catalyzes site-specific recombination between an attachment site on the infecting phage chromosome (attP) and an attachment site in the host chromosome (attB) in natural system. The resulting integration reaction inserts the phage genome into the host chromosome bracketed by newly formed attL and attR (LR) sites. When attB and attP are engineered to be opposite BP sites, the integrase alone catalyzes the inversion of sequences flanked by BP sites, changing BP sites into LR sites, and will not revert the DNA flanked by LR sites.

In the design of Eu-timer (Fig 1) , each inverted promoter flanked by BP sites is downstream of an inverted reporter gene and followed by a ser integrase gene. The reporter i(inverted reporter gene)-attP-promoter i-attB-integrase unit is defined as a counting motif, named eu-timer integrase motif (EIM).

The circuit can be programmed to record time by counting a specific type of events like the expression of cyclins. Once the motifs are activated, the downstream expression can work automatically and will not be terminated or reset by the hosts themselves, which is the reason why we believe that such a system can imprint “the same time” on microbes derived from a single clone.

We then designed an telomere-like device by making little changes upon Micro timer 1.0, named Micro timer 1.1 (Fig 2) , in which the flanking site are of same direction. With every cell division, this device will sequentially truncate a part of the sequence, and finally lead to cell death, working like telomere.