Part:BBa_K5351005

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]

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

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

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

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

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)
(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

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)
(a: XI-2-XI colonies; b: Validation of colony PCR of XI-2-PsXI; c: Sequencing result of XI-2-XI)

3. X-3-2XI

We constructed a plasmid X-3-2XI containing two copies of the XI genes. Figure 8 shows the PCR validation results for the promoter GAP, the coding gene PsXI, and the terminator CYC1, with band sizes matching the expected lengths of 693 bp, 1350 bp, and 275 bp, respectively, indicating successful amplification.

Figure 8. Amplification result of GAP pro, PsXI, and CYC1 ter

Overlap PCR was performed on these fragments, and Figure 9 shows the results of the overlap PCR, with a band size consistent with the expected length of 2245 bp, indicating successful synthesis of the target gene.

Figure 9. Overlap PCR result of GAP-PsXI-CYC1

We amplified and validated the backbone X-3-XI and the target gene GAP-PsXI-CYC1. The results in Figure 10 showed matching band sizes, indicating successful amplification.

Figure 10. Validation of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the X-3-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli DH5α. Figure 11a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies, and Figure 11b displays the results of the colony PCR, showing bands of approximately 904 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 11c, the GAP-PsXI-CYC1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the X-3-2XI plasmid.

Figure 11. The Monoclonal antibody validation and sequencing of X-3-2XI (DH5α)
(a: X-3-2XI colonies; b: Validation of colony PCR of X-3-2XI; c: Sequencing result of X-3-2XI)

4. XI-2-2XI

We constructed a plasmid XI-2-2XI containing two copies of the XI genes. We amplified and validated the backbone XI-2-XI and the target gene GAP-PsXI-CYC1. The results in Figure 12 showed matching band sizes, indicating successful amplification.

Figure 12. Validation result of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the XI-2-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli DH5α. Figure 13a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies, and Figure 13b displays the results of the colony PCR, showing bands of approximately 904 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 13c, the GAP-PsXI-CYC1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the XI-2-2XI plasmid.

Figure 13. The Monoclonal antibody validation and sequencing of XI-2-2XI (DH5α)
(a: XI-2-2XI colonies; b: Validation of colony PCR of XI-2-2XI; c: Sequencing result of XI-2-2XI)