Coding

Part:BBa_K5351005

Designed by: Aimi Ma   Group: iGEM24_FDfzSH   (2024-09-02)
Revision as of 10:41, 27 September 2024 by Baldeep (Talk | contribs)

PsXI



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 910
  • 1000
    COMPATIBLE WITH RFC[1000]

BBa_K5351005 Documentation

Origin

Saccharomyces cerevisiae; synthesized

Properties

Xylose isomerase (XI) is an enzyme responsible for catalyzing the isomerization of xylose to xylulose, an essential step in the metabolism of xylose.

Usage and Biology

Xylose isomerase (XI) is an enzyme responsible for catalyzing the isomerization of xylose to xylulose, an essential step in the metabolism of xylose. This enzyme is crucial in microbial strains engineered for xylose utilization, as it enables the conversion of xylose into a form that can enter the pentose phosphate pathway for energy production. XI plays a crucial role in enhancing the efficiency of xylose utilization in various biotechnological applications, such as biofuel production and metabolic engineering of microorganisms for the conversion of lignocellulosic biomass into valuable products.

Figure 1. Gene map of PsXI
Figure 1. Gene map of PsXI

Experimental Approach

1. X-3-XI

We constructed a plasmid X-3-XI containing a single copy of the XI gene. Figure 2 shows the PCR validation results for the promoter TEF1, the coding gene PsXI, and the terminator ADH1, with band sizes matching the expected lengths of 430 bp, 1354 bp, and 214 bp, respectively, indicating successful amplification.

Figure 2. Amplification result of TEF1 pro, PsXI, and ADH1 ter
Figure 2. Amplification result of TEF1 pro, PsXI, and ADH1 ter

Overlap PCR was performed on these fragments. Figure 3 shows the results of the overlap PCR, with a band size consistent with the expected length of 1920 bp, indicating successful synthesis of the target gene.

Figure 3. Overlap PCR result of TEF1-PsXI-ADH1
Figure 3. Overlap PCR result of TEF1-PsXI-ADH1

We amplified and validated the backbone X-3 and the target gene TEF1-PsXI-ADH1. The results in Figure 4 showed matching band sizes, indicating successful amplification.

Figure 4. Validation of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1
Figure 4. Validation of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1

We ligated the X-3 backbone and the target gene TEF1-PsXI-ADH1 and transformed it into competent E.coli DH5α. Figure 5a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies, and Figure 5b displays the results of the colony PCR, showing bands of approximately 2065 bp, consistent with the expected fragment size, validating our successful transformation and plasmid construction.

The colonies were also sent for sequencing. According to the results shown in Figure 5c, the TEF1-PsXI-ADH1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the X-3-XI plasmid.

Figure 5. The Monoclonal antibody validation and sequencing of X-3-XI (DH5α)
Figure 5. The Monoclonal antibody validation and sequencing of X-3-XI (DH5α)
(a: X-3-XI colonies; b: Gel electrophoresis validation of colony PCR of X-3-2XI colonies; c: Sequencing result of X-3-2XI)

2. XI-2-XI

We constructed a plasmid XI-2-XI containing a single copy of the XI gene. We amplified and validated the backbone XI-2 and the target gene TEF1-PsXI-ADH1. The results in Figure 6 showed matching band sizes, indicating successful amplification.

Figure 6. Validation result of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1
Figure 6. Validation result of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1

We ligated the XI-2 backbone and the target gene TEF1-PsXI-ADH1 and transformed it into competent E.coli DH5α. Figure 7a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies, and Figure 7b displays the results of the colony PCR, showing bands of approximately 2065 bp, consistent with the expected fragment size, validating our successful transformation and plasmid construction.

The colonies were also sent for sequencing. According to the results shown in Figure 7c, the TEF1-PsXI-ADH1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the XI-2-XI plasmid.

Figure 7. The Monoclonal antibody validation and sequencing of XI-2-XI (DH5α)
Figure 7. The Monoclonal antibody validation and sequencing of XI-2-XI (DH5α)
(a: XI-2-XI colonies; b: Validation of colony PCR of XI-2-PsXI; c: Sequencing result of XI-2-XI)

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