Plasmid

Part:BBa_K4873054

Designed by: Qiu Jiaying   Group: iGEM23_Shanghai-city   (2023-09-24)


pUCzli-SacB-RFPT


iGEM Team Contribution

Composite Part: BBa_K4873054 (pUCzli-SacB-RFPT)

Construction Design

We constructed a new combinatorial plasmid BBa_K4873054 (pUCzli-SacB-RFPT). It is composed of BBa_K4873052 (pUCzli-SacB) and BBa_K4873006 (RFP). The pUCzli-SacB includes the zliE-zliS regulatory system (including promoter and terminator), which was cloned into the vector pUC19, and then the sucrase sacB gene was inserted into pUCzliES to construct the plasmid pUCzli-SacB. Finally, RFP was homologously recombined into pUCzli-SacB.

Figure 1
Figure 1. The construction of pUCzli-SacB-RFPT

Engineering Principle

In prokaryotes, levansucrase (SacB) can decompose sucrose into glucose and fructose. This process is completed by levansucrase1. Levansucrase SacB is an important protein that decomposes sucrose to produce levan. Levansucrase SacB, derived from Zymomonas mobilis, is an important protein that decomposes sucrose to produce levan. The expression and transport of this enzyme are regulated by the functional genes zliE and zliS. We constructed pUCzli-SacB, which is composed of ZliE-zliS and SacB2-3. On this basis, we added RFP to use the fluorescence intensity of RFP to characterize the feasibility of the system. The levansucrase gene was fused with the RFP reporter gene, and the fluorescence of RFP was combined with the yield of levan to characterize the colorless metabolites without reliable screening phenotype2-3.

Experimental Approach

  1. We successfully obtained the target fragment, such as zliE-zliS, promoter, SacB, RFP, through PCR amplification. As shown in Figure 2, we confirmed their consistent sizes using DNA gel electrophoresis.
    Figure 2
    Figure 2. Analysis of DNA gel electrophoresis for the identification of the target fragment
  2. Construction of plasmid 1 (pUC-zliES)

    In order to construct plasmid 1, we used PCR to amplify the EV (empty vector) pUC19 and zliES. Then we used enzyme digestion to cut a same shape of the end of the DNA of pUC19 and zliES respectively. Finally, they were combined together by T4-DNA ligase (Figure 3).

    Figure 3
    Figure 3. Monoclonal cultivation and sequencing results indicate the successful construction of pUC-zliES

  3. Construction of plasmid 2 (pUC-zli-SacB)

    By using PCR to amplify SacB and IPCR to amplify plasmid 1, we had successfully obtained recombinant plasmid. Afterwards, we still used enzyme digestion to cut the same shape of the end of SacB and plasmid 1. Through Figures 4 and 5, we have successfully constructed pUC-zli-SacB.

    Figure 4
    Figure 4. Monoclonal cultivation and identification of the SacB fragment after enzyme digestion through DNA gel electrophoresis
    Figure 5
    Figure 5. Sequencing results indicate the successful construction of pUC-zli-SacB

  4. Construction of plasmid 3 (pUC-zli-SacB-RFP)

    Similar to the previous sections, we used PCR to amplify plasmid 2 and RFP, while instead of using enzyme digestion and ligation, we used homologous recombination to combine plasmid 2 and RFP. As shown in Figures 6 and 7, we have successfully constructed pUC-Zli-SacB-RFP.

    Figure 6
    Figure 6. Identification of the RFP fragment after enzyme digestion through DNA gel electrophoresis
    Figure 7
    Figure 7. Sequencing results indicate the successful construction of pUC-Zli-SacB-RFP

Reference:

  1. Liu L, et al. The potential use of Zymomonas mobilis for the food industry. Crit Rev Food Sci Nutr. 2022, 10.1080/10408398.2022.2139221
  2. Dai Y, et al. Lowering whole cost for sugarcane based ethanol production by engineered Zymomonas mobilis. GCB Bioenergy, 2021, 13(12), 1894-1907.
  3. Kondo Y, et al., Cloning and characterization of a pair of genes that stimulate the production and secretion of Zymomonas mobilis extracellular levansucrase and invertase. Bioscience, Biotechnology, and Biochemistry 1994, 58:3, 526-530


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 415
    Illegal XhoI site found at 3215
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3630
    Illegal AgeI site found at 6148
    Illegal AgeI site found at 6260
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1751
    Illegal BsaI.rc site found at 3097
    Illegal SapI site found at 668


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