Plasmid

Part:BBa_K5348023

Designed by: ER DU   Group: iGEM24_Songshan-Lake   (2024-09-03)
Revision as of 13:05, 30 September 2024 by Baldeep (Talk | contribs)

_ pYC-pKC-pL-RBS2-MazF



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 5847
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4560
    Illegal NgoMIV site found at 4632
    Illegal NgoMIV site found at 4722
    Illegal NgoMIV site found at 4740
    Illegal NgoMIV site found at 5232
    Illegal NgoMIV site found at 5525
    Illegal NgoMIV site found at 5619
    Illegal AgeI site found at 4274
    Illegal AgeI site found at 5400
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5289
    Illegal BsaI.rc site found at 4173
    Illegal SapI site found at 1
    Illegal SapI.rc site found at 3967


pYC-pKC-pL-RBS3-MazF (BBa_K5348024)

pYC-pKC-pL-RBS3-MazF (BBa_K5348024)

Summary

To reduce the leaky expression of the light-on induced system (BBa_K3447133), we reduced the strength of the RBS, which is connected to the target genes. We tested the light-controlled regulation of the toxin protein MazF as a model protein, and the experimental results showed that we successfully realized the regulation of bacterial growth through this composite part.

Construction Design

This composite part consists of the pL-RBS3 (BBa_K5348006), toxin protein MazF (BBa_K1096002), and pTrc99k-backbone (BBa_K3999002). With the pL light-control system, we hope to regulate MazF expression in the dark and under blue light as a way to control bacterial growth.

Figure 1. Schematic diagram of pYC-pKC-pL-RBS3-MazF
Figure 1. Schematic diagram of pYC-pKC-pL-RBS3-MazF

Engineering Principle

The pL light-control system consists of several basic parts. Under dark condition, histidine kinase (YF1) phosphorylates FixJ (response regulator of histidine kinase), which activates PFixK2 (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, PR (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, PR can be transcribed normally, and downstream genes can be expressed [1].

Experimental Approach

The plasmid construction scheme is shown in Figure 2A. We amplified the pL-1, pL-2-RBS(3), RBS(3)-MazF, and pTrc99k backbone fragments, respectively. To improve the efficiency of homologous recombination, we first used overlap PCR to obtain the pL-2-RBS(3)-MazF fragment, and then we homologously recombined the pL-1, pL-2-RBS(3)-MazF, and pTrc99k backbone fragments. Colony PCR and sequencing results confirmed the successful construction of the pYC-pKC-pL-RBS(3)-MazF plasmid (Figure 2B).

Figure 2. Construction results of pYC-pKC-pL-RBS(3)-MazF plasmid
Figure 2. Construction results of pYC-pKC-pL-RBS(3)-MazF plasmid. (A) Construction Strategy. (B) Colony PCR and sequencing results.

Measurement: Light Control Test

We conducted light-control tests on the successfully constructed strains. Results showed that under blue light cultivation, pL-RBS(3)-MazF reduced bacterial concentration (OD600) by 1.2 times compared to dark conditions. This indicates that under blue light, the toxic protein MazF was successfully expressed and inhibited bacterial growth, demonstrating that the pL element can regulate MazF expression (Figure 3).

Figure 3. Light-control tests results
Figure 3. Light-control tests results.

References

[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.

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