Part:BBa_K2447501

Blue light activated inducible system with RFP reporter

In this construct, the photosensitive bacterial protein is constructively produced. In the presence of blue light, EL222 is activated via dimerisation and able to bind upstream of the luxI promoter, thereby allowing the recruitment of RNA polymerase to elicit downstream expression of RFP. In the absence of blue light, EL222 does not dimerise and unable to bind to DNA; hence eliciting minimal RFP expression.

Figure 1: Under blue-light condition, EL222 is recruited upstream of the RNA polymerase binding region and recruits the polymerase for downstream expression of RFP. Figure is obtained from this paper.

Usage and Characterisation

This part is inserted into pBbE8k backbone and subsequently characterised in E.coli Beta 10 and E.coli MG 1655. These cells are grown in LB medium (with kanamycin) at 37 degree to reach an OD of 0.1 before blue light administered for 7 hours. For every hour, RFP readings were obtained and cross-compared with the control (dark condition).

Figure 2: Incubator-shaker used in the blue light experiment.

Figure 3: Blue light being shone on one plate while no light condition was stimulated with the covering of a black cloth.

Strong RFP expressions were elicited from both cell types (under blue-light condition) as compared to the control condition where no light was administered. By the 6th hour onwards, RFP productions had tripled for both cell types when compared to the control condition In short, this construct could prove useful for any group seeking to elucidate gene expression based on blue light as a physical stimulus.

Figure 4: MG 1655 under blue light and no light conditions.

Figure 5: Beta 10 under blue light and no light conditions.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 527
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 97
  Illegal AgeI site found at 1473
  Illegal AgeI site found at 1585
  
• 1000
  COMPATIBLE WITH RFC[1000]