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 is able to bind upstream of the pLuxI promoter, thereby allowing the recruitment of RNA polymerase to activate expression of RFP. In the absence of blue light, EL222 does not dimerise and is unable to bind DNA; hence showing 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 article.

Usage and Characterisation

This part is inserted into pBbE8k backbone and subsequently characterized in E. coli 10Beta and MG 1655. These cells are grown in LB medium (with kanamycin) at 37 degrees to reach OD600 of 0.1 before blue light is administered for 7 hours. For every hour, RFP readings were obtained and cross-compared with the control (no blue light).

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

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

Strong RFP expression was shown 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: 10Beta under blue light and no light conditions.

Characterization by WHU-China 2023

We synthesized this composite part and incorporated it into pET-28a. However, in our experiments, it can’t work as expected. There is little difference between the expression of mRFP1 before and after induction in E.coli BL21 with pET-28a-[EL222-mRFP1] (Fig1). After comparing the sequence with other EL222 protein parts, we found a deletion in the sequence of EL222 from this part (Fig2). In fact, because of the lack of initiation codon, EL222 proteins cannot be produced in bacteria. Based on these facts we found, we designed a new composite part <a href="http://https://parts.igem.org/Part:BBa_K4630301">BBa_K4630301</a>, and achieved our own characterization.

Figure 1:Image of mRFP expression in E.coli BL21 with pET-28a-[EL222-mRFP1]

Figure 2:Sequence Alignment of EL222

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]