Part:BBa_K4284022

pCDFD-bktB-ydil

pCDFD-bktB-ydil

contribution

The gene ydiI (Gene ID: 946190), which is also known as menl, works as 1,4-dihydroxy-2-naphthoyl-CoA hydrolase, and it is conserved in the E. coli MG1655 genome. The E. coli thioesterase YdiI was used as the cycle-terminating enzyme, as it was found to have not only the ability to convert trans-enoyl-CoAs to the corresponding α, β-UCAs, but also a very low catalytic efficiency on acetyl-CoA, the primer and extender unit for the r-BOX pathway. In our project, we overexpressed this enzyme by cloning it into the plasmid pCDFDeut1 to produce MCFAs.

pCDFD-bktB-ydiI is a composite part that contains the key enzymes bktB and ydiI. The plasmid backbone pCDFDeut-1 is usually used for protein expression. It is designed for the co-expression of two target ORFs, and this carrier contains two multiple clone sites, and each site has a T7-lac promoter and a ribosome binding site (RBS).

The gene bktB is a β-ketothiolases that play a key role in the r-BOX cycle and is utilized in the in vivo conversion of Coenzyme A (CoA)-linked precursors such as acetyl-CoA and glycolyl-CoA into β-hydroxy acids.

Figure 1. Construction of pCDFD-bktB-t7ydil..

Engineering Success

a) Construction of transcriptional activation screening platform

In order to construct our plasmids, we amplify all four enzymes with corresponding templates by PCR. the plasmid pUC57-bktb was used as the template to amplify gene bktB, and the genome of E. coli MG1655 was used as the template to amplify gene ydiI (Figure 2).

Figure 2. Gel electrophoresis results of target gene fragments. Line 1: DNA marker Line 2: bktB 1176bp Line 3: ydiI 501bp..

In Figure 2 we can find that there were four clear DNA bands, indicating that genes bktB, ydiI, were successfully amplified by PCR.

Meanwhile, we also used a one-step cloning method to ligate the gene bktB into the NcoI and BamHI sites of the pCDFDuet-1 vector. Then we transformed the recombinant plasmid into E. coli DH5α competent cells. We inoculated the correct strain and extracted the plasmid pCDFDuet1-bktB. Then we inserted gene ydiI into the NdeI and XhoI sites of pCDFDuet1-bktB through a one-step cloning method. The recombinant plasmid pCDFDuet1-bktB-ydiI was verified by NcoI/HindIII and NdeI/XhoI, respectively (Figure3 line1, 3).

Figure 3. Digestion verification of pETDuet1-fadB-ter and pCDFDuet1-bktB-ydiI. M: DNA marker 3: ter fragment and carrier fragment 4: bktB fragment and carrier fragment..

Figure 4. The map of pCDFDuet1-bktB-ydiI. ..

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 5.), and the plasmids were successfully constructed. So far, we have successfully obtained the recombinant plasmids.

Figure 5. The sequencing blast results of the recombinant plasmids. 5-1. Sequencing and comparison of pCDF-bktB..

Figure 5. The sequencing blast results of the recombinant plasmids. 5-2. Sequencing and comparison of pETD-ydiI..

a) Protein expression and purification

In order to obtain the two enzymes, we transferred the recombinant plasmid, pCDFDuet1-bktB-ydiI, into E. coli BL21(DE3), inoculated the correct colony in the LB culture medium and added IPTG to induce protein expression when the OD600 reached 0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the enzymes we wanted, and detected the proteins by SDS-PAGE (Figure 6).

Figure 6. SDS-PAGE analysis of bktB, ydiI. Line 1: marker P2: bktB P3: ydiI..

Two clear protein bands can be seen in figure 6, while there is no corresponding band in the control, indicating the successful expression of bktB, ydiI.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2987
  Illegal XbaI site found at 5101
  Illegal PstI site found at 430
  Illegal PstI site found at 1069
  Illegal PstI site found at 2075
  Illegal PstI site found at 2714
  Illegal PstI site found at 3006
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2987
  Illegal NheI site found at 4301
  Illegal PstI site found at 430
  Illegal PstI site found at 1069
  Illegal PstI site found at 2075
  Illegal PstI site found at 2714
  Illegal PstI site found at 3006
  Illegal NotI site found at 3024
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2987
  Illegal BamHI site found at 2981
  Illegal XhoI site found at 3089
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2987
  Illegal XbaI site found at 5101
  Illegal PstI site found at 430
  Illegal PstI site found at 1069
  Illegal PstI site found at 2075
  Illegal PstI site found at 2714
  Illegal PstI site found at 3006
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2987
  Illegal XbaI site found at 5101
  Illegal PstI site found at 430
  Illegal PstI site found at 1069
  Illegal PstI site found at 2075
  Illegal PstI site found at 2714
  Illegal PstI site found at 3006
  Illegal NgoMIV site found at 3554
  Illegal AgeI site found at 1213
  Illegal AgeI site found at 2858
  Illegal AgeI site found at 3301
  
• 1000
  COMPATIBLE WITH RFC[1000]