Part:BBa_K750107

pBADcIT-0.07:cI protein expression system activated by arabinose(RBS strength:0.07)

Description

This part contains promoter pBAD, RBS of 0.07 strength, cI(lva) protein and double terminator. When arabinose is added, the promoter pBAD will be activated and cI(lva) protein will be produced. cI protein can activated promoter PcI, and repress the production of reporter gene behind it. In our project, sometimes we need the cell can be switched from light to dark, so we designed PciGLT(BBa_K750103), a GFP expression system which can be repressed by cI protein. And this part can provide cI protein for PcIGLT.

Performance

In our project, we utilized tubes with different kinds of bacteria as seven segments. According to the theory of network design, genetic logic gates were assembled into circuits and then constructed using synthetic biology method. Based on the knowledge of bioinformatics and synthetic biology, there are only two results for a single logic gate: on or off. Here, instead of electric signals representing streams of binary ones and zeros, the chemical concentrations of specific DNA-binding proteins and inducer molecules act as the input and output signals of the genetic logic gates, i.e. present or absent of a specific signal molecule represents binary one or zero. The genetic circuits we constructed actually using Binary-Coded Decimal (or BCD, a device which converts the binary numerical value to decimal value) to compute signal molecules.To display two results (in this case, number one and zero) necessitates one kind of signal molecule, we called it a Single-input BCD Decoder (As shown in Table 1).

Table 1. Design of circuits displaying the number 0 and 1. The inducer is arabinose, which effect on promoter PBAD (an E.coli promoter, see more[4]). In the absence of arabinose, cI regulated promoter(PcI) expresses GFP with LVA tag. In the presence of arabinose, PBAD starts transcription and then repressor CI protein is translated and represses PcI.

Table 2 presents the design of device displaying the number 0, 1, 2, which we called Dual-input BCD Decoder. Once the construction is completed, Engineered E.coli with different circuits can be immobilized into microcapsules and then be put in seven transparent tubes which acting as segment of display.

Table2.Biological digital display which responds to two inducers, displaying the number 0, 1, 2

we encountered a thorny problem that synthetic NOT gate in PBADcIT-PcIGLT (Part:BBa_K750113, short for PBAD-rbs-cI-tt-PcI-rbs-gfp(LVA)-tt) and PtetcIT-PciGLT (Part:BBa_K750114, short for Ptet-rbs-cI-tt-PcI-rbs-gfp(LVA)-tt) failed to function, which is supposed to express GFP without any inducer. However, no fluorescence can be observed or detected. This result may be explained by the possible leakage of expression of CI repressor protein. In order to verify our suspicion, we performed SDS-PAGE to detect the existent of CI protein. We use the front parts of the circuit as new parts, i.e. PBADcIT (Part:BBa_K750104, short for PBAD-rbs-cI-TT).

But we got no result from the SDS-PAGE, it is possible to hypothesize that the SDS-PAGE is not so sensitive to detect the low concentration of CI protein, since we have no time to transfer the strain from DH5α to BL21. We did further experiment using an additional group of another part with lower strength of RBS: PBADcIT-0.07 (Part:BBa_K750107 , short for PBAD-rbs0.07-cI-tt). The aim of constructing this new part is to reduce the hypothetic leakage of CI expression. If succeed in this function, the new part could then be assembled with the downstream part, functioning as genetic circuits mentioned before. Additionally, the experiment conducted on strains that had been transferred into BL21 earlier, to prevent the low expression level due to the defection of DH5α. By comparing BL21 group and the group shown in Figure 1, no leakage of CI protein can be observed, though changes in the strength of RBS are effective.

Figure1.Protein electrophoresis of PBADcIT with RBS0.07 and RBS1.0. S1: BL21; S2: Control of PBADcIT with RBS0.07; S3: PBADcIT with RBS0.07 induced by 1.0 mM arabinose; S4: Control of PBADcIT with RBS1.0; S5: PBADcIT with RBS1.0 induced by 1.0 mM arabinose.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]