Coding

Part:BBa_K4842001

Designed by: Niu MuYao   Group: iGEM23_WestCentral-HS   (2023-08-08)
Revision as of 09:35, 11 October 2023 by Cey0616 (Talk | contribs)


pnar

pnar

New part: Pnar

Name: Pnar

Part: BBa K4842001 - parts.igem.org

Base Pairs: 237 bp

Origin: Escherichia coli str. K-12 substr. MG1655

Properties: Promoter

Usage and Biology

Despite some exciting achievements in the heterologous biosynthesis of (S)-estragenol, heterologous expression of this pathway in E. coli currently relies on isopropyl-β-d-thiogalactopyranoside (IPTG) induction, and the high price and toxicity of IPTG to E. coli cells have limited the application of this induced expression on an industrial scale[1]. The dissolved oxygen (DO)-dependent nar promoter is considered an alternative to the above promoters because of its relatively simple and cost-effective induction mechanism and function at any stage of cell growth[2]. Therefore, our hypothesis was whether this oxygen-responsive promoter could be used to control our (S)-estradiol biosynthesis pathway in aerobic anaerobic two-stage cultures to achieve access to green-produced (S)-estradiol in a cost-effective manner. In addition, our nar promoter is derived from a 1992 reference [3] with the sequence name pMW618. It should be noted that the promoter ontology is 197 bp in length, and to facilitate the study, our sequence also contains enzymatic and RBS sites totaling 237 bp.

Experimental Approach

1. Construction of plasmid pETM6-pnar

To construct the plasmid pETM6-pnar, we first double-digested the synthetic pCDM4-pnar and pETM6 with NdeI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the nar fragment with the pETM6 vector using T4 DNA ligase to obtain the complete recombinant plasmid.

Figure 1: Construction result of the plasmid pETM6-pnar.

Figure 1: Construction result of the plasmid pETM6-pnar.

2. Construction of plasmid pETM6-pnar-mCherry

To construct plasmid pETM6-pnar-mCherry, we first double-digested pET28a-mCherry and pETM6-pnar with XhoI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the mCherry fragment with the pETM6-pnar vector using T4 DNA ligase to obtain the complete recombinant plasmid.

Figure 2: Construction result of the plasmid pETM6-pnar-mCherry.

Figure 2: Construction result of the plasmid pETM6-pnar-mCherry.

Characterization/Measurement

Mutation library construction for the plasmids pETM6-pnar-RBSx-mCherry

We designed eight RBS sequences downstream of the nar promoter using the RBS Calculator (https://salislab.net/software/), as shown in the table below.

Table 1: RBS sequences designed by RBS Calculator

Table 1: RBS sequences designed by RBS Calculator.

We then introduced the RBS sequence mutations by whole plasmid PCR. We successfully amplified pETM6-pnar-RBS(1-8)-mCherry plasmids containing different RBS sequences.

Figure 3: Electrophoresis results of whole plasmid PCR.

Figure 3: Electrophoresis results of whole plasmid PCR.

We transformed pETM6-pnar-RBS (1-8)-mCherry plasmids into competent E. coli BL21(DE3) cells, respectively. After overnight culture, positive transformants grew on LB plates. Colony PCR identification showed the correct bands for pETM6-pnar-RBS (1-5)-mCherry transformants, while no bands were observed for pETM6-pnar-RBS (6-8)-mCherry. This may be due to mistakes in picking colonies or omissions in adding templates. Nevertheless, we inoculated all eight transformants for subsequent fluorescence intensity testing.

Figure 4: Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry.

Figure 4: Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry.

Reference

  1. Lee P G, Kim J, Kim E J, et al. P212A mutant of dihydrodaidzein reductase enhances (S)-equol production and enantioselectivity in a recombinant Escherichia coli whole-cell reaction system[J]. Applied and Environmental Microbiology, 2016, 82(7): 1992-2002.
  2. Clomburg J M, Blankschien M D, Vick J E, et al. Integrated engineering of β-oxidation reversal and ω-oxidation pathways for synthesizing medium chain ω-functionalized carboxylic acids[J]. Metab Eng, 2015, 28(202-212).
  3. Walker, M.S., DeMoss, J.A., 1992. Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli. J Bacteriol 174(4), 1119-1123.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 207
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 207
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 207
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 207
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 207
  • 1000
    COMPATIBLE WITH RFC[1000]


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