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.