Contribution
1.Add new basic parts, BBa_K5351000(pSCm-N20); BBa_K5351002(NFS1mu); BBa_K5351003(ISU1); BBa_K5351005(PsXI); BBa_K5351010(X-3-XI-2-gRNA-Backbone)
1.1 BBa_K5351000(pSCm-N20)
Fig 1. Plasmid map of pSCm-N20
1.2 BBa_K5351002(NFS1mu)
Fig 2. Gene map of NFS1
1.3 BBa_K5351003(ISU1)
Fig 3. Gene map of ISU1
1.4 BBa_K5351005(PsXI)
Fig 4. Gene map of PsXI
1.5 BBa_K5351010(X-3-XI-2-gRNA-Backbone)
Fig 5. Plasmid map of X-3-XI-2-gRNA-Backbone
2.Add new composite parts, BBa_K5351001(pSCm-NFS1mu); BBa_K5351004(pSCm-ISU1); BBa_K5351006(X-3-XI); BBa_K5351008(XI-2-XI); BBa_K5351007(X-3-2XI); BBa_K5351009(XI-2-2XI)
2.1 BBa_K5351001(pSCm-NFS1mu)
The gRNA plasmid pSCm-NFS1mu (BBa_K5351001) is designed for the yeast strain where the NFS1 (BBa_K5351002) gene is mutated. The critical gene in the plasmid is the NFS1 gRNA, combined with the SNR52 promoter and SUP4 terminator. A mutation will later be induced on the NFS1 gene in the yeast to promote the metabolism of xylose. It is constructed by connecting two N20 oligos and the gRNA backbone.
We constructed pSCm-NFS1mu by ligating the pSCm-N20 (BBa_K5351000) backbone and the target gene NFS1. The pSCm was digested to obtain a linear backbone with a length of 5984 bp. Fig 6 indicates the band is consistent with the results.
Fig 6. The gel electrophoresis validation of the linear pSCM-N20 plasmid backbone.
These backbones were ligated with the target gene NFS1 and transformed into competent E.coli DH5α. Fig 7a shows the presence of single colonies on the plate. Colony PCR was performed to validate the colonies. Fig 7b displays the results of colony PCR, showing bands of 288 bp, consistent with the expected length, thus validating our successful plasmid construction.
The colonies were also sent for sequencing. According to the results shown in Fig 7c, the NFS1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the pSCm-NFS1 plasmid.
Fig 7. The Monoclonal antibody validation and sequencing of pSCm-NFS1 (DH5α) (a: pSCm-NFS1 colonies; b: the gel electrophoresis validation of colony PCR of pSCm-NFS1; c: sequencing result of pSCm-NFS1)
2.2 BBa_K5351004(pSCm-ISU1)
The key gene in the pSCm-ISU1(BBa_K5351004) plasmid is ISU1 gRNA, with SNR52 promoter and SUP4 terminator. It is constructed by connecting two N20 oligo and the gRNA backbone. We generated pSCm-ΔISU1 through the ligation of the pSCm backbone with the target gene ISU1(BBa_K5351003).
We generated pSCm-ΔISU1 through the ligation of the pSCm backbone with the target gene ΔISU1, followed by transformation into competent E. coli DH5α. Fig 8a illustrates the presence of individual colonies on the plate.
A colony PCR was conducted to validate the colonies. The results are depicted in Fig 8b, which exhibits bands of 288 bp, aligning with the expected length, verifying the successful construction of the plasmid.
Furthermore, sequencing was performed on the colonies. As demonstrated in Fig 8c, the ΔISU1 gene was seamlessly ligated to the backbone without any detectable mutations, confirming the accurate construction of the pSCm-ΔISU1 plasmid.
Fig 8. The Monoclonal antibody validation and sequencing of pSCm-ΔISU1 (DH5α) (a: pSCm-ΔISU1 colonies; b: the gel electrophoresis validation of colony PCR of pSCm-ΔISU1 colonies; c: sequencing result of pSCm-ΔISU1)
2.3 BBa_K5351006(X-3-XI)
X-3-XI(BBa_K5351006) is constructed by integrating the PsXI (BBa_K5351005) gene into the X-3(BBa_K4845003) site. A biotech company provides the backbone plasmid of the X-3 integration site. The TEF1 promoter(BBa_K4703017) and ADH1 terminator(BBa_K3803006) join the PsXI gene. We constructed a plasmid X-3-XI containing a single copy of the XI gene. Fig 9 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.
Fig 9. Amplification result of TEF1 pro, PsXI, and ADH1 ter
Overlap PCR was performed on these fragments, as shown in Fig 10, which 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.
Fig 10. 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 Fig 11 showed matching band sizes, indicating successful amplification. We ligated the X-3 backbone and the target gene TEF1-PsXI-ADH1.
Fig 11. 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. Fig 12a shows the results after culturing E. coli, where single colonies can be observed.
We performed colony PCR to validate the cultured colonies. Fig 12b 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 Fig 12c, 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.
Fig 12. The Monoclonal antibody validation and sequencing of X-3-XI (DH5α) (a: X-3-XI colonies; b: the gel electrophoresis validation of colony PCR of X-3-2XI colonies; c: sequencing result of X-3-2XI)
2.4 BBa_K5351008 (XI-2-XI)
We constructed a plasmid XI-2-XI (BBa_K5351008) containing a single copy of the XI (BBa_K5351005) gene. The integration site is XI-2(BBa_K4845002). We amplified and validated the backbone XI-2 and the target gene TEF1-PsXI-ADH1. The results in Fig 13 showed matching band sizes, indicating successful amplification. We ligated the XI-2 backbone and the target gene TEF1-PsXI-ADH1.
Fig 13. 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. Fig 14a shows the results after culturing E. coli, where single colonies can be observed.
We performed colony PCR to validate the cultured colonies. Fig 14b 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 Fig 14c, 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.
Fig 14. 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)
2.5 BBa_K5351007 (X-3-2XI)
We constructed a plasmid X-3-2XI (BBa_K5351007) containing two copies of the XI genes. The integration site is X-3(BBa_K4845003). Fig 15 shows the PCR validation results for the promoter GAP(BBa_K4845004), the coding gene PsXI(BBa_K5351005), and the terminator CYC1(BBa_K4000002), with band sizes matching the expected lengths of 693 bp, 1350 bp, and 275 bp, respectively, indicating successful amplification.
Fig 15. Amplification result of GAP pro, PsXI, and CYC1 ter
Overlap PCR was performed on these fragments, as shown in Fig 16, which 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.
Fig 16. Overlap PCR result of GAP-PsXI-CYC1
We amplified and validated the backbone X-3-XI(BBa_K5351006) and the target gene GAP-PsXI-CYC1. The results in Fig 17 showed matching band sizes, indicating successful amplification. We ligated the X-3-XI backbone and the target gene GAP-PsXI-CYC1
Fig 17. 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. Fig 18a shows the results after culturing E. coli, where single colonies can be observed.
We performed colony PCR to validate the cultured colonies. Fig 18b 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 Fig 18c, 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.
Fig 18. 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)
2.6. BBa_K5351009(XI-2-2XI)
We constructed a plasmid XI-2-2XI(BBa_K5351009) containing two copies of the XI (BBa_K5351005) genes. The integration site is XI-2(BBa_K4845002). We amplified and validated the backbone XI-2-XI and the target gene GAP-PsXI-CYC1. The results in Fig 19 showed matching band sizes, indicating successful amplification. We ligated the XI-2-XI backbone and the target gene GAP-PsXI-CYC1.
Fig 19. validation result of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1
We ligated the XI-2-XI(BBa_K5351008) backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli DH5α. Fig 20a shows the results after culturing E. coli, where single colonies can be observed. We performed colony PCR to validate the cultured colonies. Fig 20b 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 Fig 20c, 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.
Fig 20. 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)