Part:BBa_K5348004

pL-RBS1

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1874
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 587
Illegal NgoMIV site found at 659
Illegal NgoMIV site found at 749
Illegal NgoMIV site found at 767
Illegal NgoMIV site found at 1259
Illegal NgoMIV site found at 1552
Illegal NgoMIV site found at 1646
Illegal AgeI site found at 301
Illegal AgeI site found at 1427
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1316
Illegal BsaI.rc site found at 200

<!DOCTYPE html> BBa_K5348004 (pL-RBS1)

BBa_K5348004 (pL-RBS1)

Summary

To reduce the leaky expression of this part, we reduced the strength of the RBS by 9-fold, which is connected to the target genes. We tested the fluorescent protein mCherry as model protein.

Construction Design

To reduce the leaky expression of the light-inducible induction system (BBa_K3447133, hereafter referred to as the pL), we reduced the intensity of the RBS linked to the target gene in this element by 9-fold (RBS1). The composition of this element is shown below.

Figure 1. Schematic diagram of the pL-RBS1

Engineering Principle

Under dark condition, histidine kinase (YF1) phosphorylates FixJ (response regulator of histidine kinase), which activates P_FixK2 (the target gene for transcription upon FixJ activation), driving the expression of the cI gene (λ phage repressor), which represses the transcription of its cognate promoter, P_R (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, P_R can be transcribed normally, and downstream genes can be expressed [1].

Experimental Approach

The plasmid construction scheme is shown in Figure 2A. We synthesized the pL element at GenScript and divided it into two fragments, pL-1 and pL-2, for synthesis. We amplified pL-1, pL-2-RBS(1) and RBS(1)-mCherry fragments, and then ligated the pL-2-RBS(1) and RBS(1)-mCherry fragments by overlapping PCR to obtain pL-2-RBS(1)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(1)-mCherry fragments, and pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(1)-mCherry plasmid (Figure 2B).

Figure 2.Construction results of pYC-pKC-pL-RBS(1)-mCherry plasmid

Measurement: Light Control Test

Subsequently, we conducted light-control tests on the strain containing pYC-pKC-pL-RBS(1)-mCherry plasmid. We cultured the strains under dark condition and blue light irradiation, respectively, sampling at intervals to measure the RFU (relative fluorescence units) of the bacterial suspension. As shown in Figure 3, the test results verified that the pL light-control element could regulate mCherry expression under dark and blue light conditions. As the RBS strength decreased, the RFU of mCherry decreased accordingly, indicating that the RBS replacement strategy can achieve regulation of the pL light-control system.

Figure 3.Light-control test results.

Reference

[1] H, Mays RL, Hoffman SM, Avalos JL. Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synth Biol. 2021 Aug 20;10(8):2015-2029.