Team:Chiba/System 1/Result

From 2010.igem.org

Requirement of realizing genetic double click system

1. T7 RNAP pulse in response to 1st and 2nd input
In response to 1st and 2nd input, T7 RNAP has to express as pulse.
To accomplish this, Lux/CI434 hybrid promoter should be activated or repressed stringently.
So, we checked this part (Data shown below).

2. CI repression
CI needs to work as a repressor of T7/CI-OR1 hybrid promoter at 1st input and needs to have degradated before 2nd input not to work as a repressor at 2nd input. To accomplish this, T7/CI-OR1 hybrid promoter should be activated or repressed stringently.
So, we checked this part (Data shown below).

3. Setting the fixed time
There is the fixed time between 1st and 2nd input. To accomplish this, CI has to re-express after washout. (Data not shown)

1-1. Lux/CI434 hybrid promoter

In detail

Hybrid promoter Design

We got the idea that cI and cI434 repressor don’t crosstalk[1], so we construct Pt7/cI and pLux/cI434 hybrid promoter.

design

1. pLux/cI434 hybrid promoter

Rationale

This hybrid promoter is designed to activated by LuxR when it is bound to AHL and repressed by the phage 434 cI repressor. The operator site sequence come from the biobrick part (BBa_R0052) and we set OR2(ttacaatgtatcttg) between -35 and -10, OR1(ttacaaactttcttg) at downstream of -10. There are only 16 bp between the -10 and -35 boxes we have confirmed that lux promoter works naturally while repression is under observation.

References and links

[1] http://web.mit.edu/synbio/x/htdocs/BB_Parts/BBa_R0050/Part.htm
[2] http://partsregistry.org/Part:BBa_I12040
[3] Basu S, Mehreja R, Thiberge S, Chen MT and Weiss R (2004). Spatiotemporal 

    control of gene expression with pulse-generating networks. Proc Natl Acad Sci USA 101:

    6355-6360.

1-2. Pulse generator

• [http://partsregistry.org/wiki/index.php?title=Part:BBa_K396006 BBa_K396006]

2. T7/CI-OR1 hybrid promoter

• [http://partsregistry.org/wiki/index.php?title=Part:BBa_K396000 BBa_K396000]

T7/CI-OR1 hybrid promoter was designed to be activated by T7 RNA Polymerase and repressed by lambda CI protein. In sequence design, lambda CI operator site 1 (OR1, Part of BBa_R1051) was directly attached to the downstream of T7 promoter sequence (BBa_I719005). This characterization was implemented to validate the promoter function. In results, the activation and repression was actually observed.

Checking the parts

Fig. How to check the parts

Fig. Data: Under the situation of CI non-expressed , T7 RNAP causes GFP expression by activating T7/CI hybrid promoter (Fig.1). On the other hand, under the situation of CI expressed, T7 RNAP does not cause GFP expression (Fig.2). The reason of this is considered as following. Under the situation of CI expressed, even though T7 RNAP tries to activate T7/CI promoter, the promoter Is repressed by CI because the ability of CI repression is superior to the one of T7 activation for this promoter. So it is concluded that T7/CI hybrid promoter is to be activated by T7 RNA Polymerase and repressed by lambda CI protein, following the rules of CI repression > T7 activation.

Reference

Dubendorf, J.W. & Studier, F.W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology 219, 45-59 (1991).