Part:BBa_K750108

PcIGLT-pBADGLT:GFP(lva) expression system controlled by cI protein or arabinose

Description

This part contains PcIGLT(BBa_K750103) and pBADGLT(BBa_K750000). The GFP from PcIGLT will be produced from the beginning. When cI protein is added to the parts, promoter PcI will be activated and repress expression of GFP. And when arabinose is added, this part will work agian because the promoter pBAD is activated. In our project, sometimes we need the cell can be switched in the order of bright-dark-bright to be used in display more numbers. So we designed this part to solve this problem.

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

When moving on to more complicate circuits in Dual-input BCD Decoder, we encountered a thorny problem. 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.

Another assumption is that the design of circuits might be unstable in E.coli, causing the totally failure of the function. Due to intrinsic uncertainties and extrinsic disturbance, this situation reported happening lot of times, much more than one can expect. To examine the circuit, a similar circuit has been constructed, where cI gene was changed into gfp gene at the downstream of pBAD. As shown in Figure 1, PcI is working and expresses GFP in the group without arabinose. For another group, after addition of arabinose, pBAD is activated and GFP in its downstream therefore start expression, resulting in a higher peak value of fluorescence. From the data, we got a preliminary result that the circuit is able to work, though without consideration about the individual difference.

Figure1.Fluorescence curves of PcIGLT-pBADGLT induced by 1 mM arabinose (red) and PcIGLT-pBADGLT without arabinose(black)

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1104
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1044
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 719
  Illegal BsaI.rc site found at 1779