Difference between revisions of "Part:BBa K523024:Design"
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By analysis of the matching CBM2 domains, it was determined that an NcoI site could easily be introduced into both at the same place without changing any amino acids. | By analysis of the matching CBM2 domains, it was determined that an NcoI site could easily be introduced into both at the same place without changing any amino acids. | ||
| − | <partinfo>BBa_K523016</partinfo> (Plac + lacZ&alpha + RBS + ''cex)'' was amplified using forward primer pSBNX3insf2 (vector-based) and a new reverse primer which introduces an NcoI site in the CBM2 domain of cex. | + | <partinfo>BBa_K523016</partinfo> (Plac + lacZα + RBS + ''cex)'' was amplified using forward primer pSBNX3insf2 (vector-based) and a new reverse primer which introduces an NcoI site in the CBM2 domain of cex. |
''cenA'' was amplified using a new forward primer which likewise introduces an NcoI site in the corresponding location of the CBM2 domain, and reverse primer pSBNX3insr2 (vector-based). | ''cenA'' was amplified using a new forward primer which likewise introduces an NcoI site in the corresponding location of the CBM2 domain, and reverse primer pSBNX3insr2 (vector-based). | ||
| − | primer cenAfNcoI: c ctg cca tgg aac ggc agc atc ccg acc | + | : primer cenAfNcoI: '''c ctg cca tgg aac ggc agc atc ccg acc''' |
| − | primer cexrNcoI: c gtt cca tgg ggc gtt gcg gac cgt cac g | + | : primer cexrNcoI: '''c gtt cca tgg ggc gtt gcg gac cgt cac g''' |
| − | The two PCR products were purified, digested with NcoI and ligated, and the ligation was used as template for PCR with primers pSB1NX2insf2 and pSB1NX3insr2. Two major products were obtained. The larger, around 3.1 kb, was presumed to be correct, and was purified from the gel. The smaller, around 2.1 kb, was presumed to be a mixture of Plac-lacZ | + | The two PCR products were purified, digested with NcoI and ligated, and the ligation was used as template for PCR with primers pSB1NX2insf2 and pSB1NX3insr2. Two major products were obtained. The larger, around 3.1 kb, was presumed to be correct, and was purified from the gel. The smaller, around 2.1 kb, was presumed to be a mixture of Plac-lacZα-''cenA'' and Plac-lacZα-''cex'' from intact template which had made it through the purification. |
The purified insert was digested with EcoRI/PstI and ligated into pSB1C3. White colonies were tested for Cex activity on MUC plates. Positive clones were miniprepped and tested for insert size. Clones were further analysed by digestion with NcoI/PstI to check that the NcoI site was present. They were then tested for CenA activity on CMC plates. | The purified insert was digested with EcoRI/PstI and ligated into pSB1C3. White colonies were tested for Cex activity on MUC plates. Positive clones were miniprepped and tested for insert size. Clones were further analysed by digestion with NcoI/PstI to check that the NcoI site was present. They were then tested for CenA activity on CMC plates. | ||
| − | It is therefore presumed, subject to confirmation of sequence, that clones which passed these tests represent the fusion of ''cex'' and ''cenA'' ( | + | It is therefore presumed, subject to confirmation of sequence, that clones which passed these tests represent the fusion of ''cex'' and ''cenA'' (i.e. Plac + lacZα + RBS + ''cxnA)'' and that this has both enzyme activities. |
===Source=== | ===Source=== | ||
Latest revision as of 18:55, 28 October 2011
cex + cenA fusion (cxnA)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 524
Illegal NotI site found at 2222 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2135
Illegal XhoI site found at 1633
Illegal XhoI site found at 1882 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 157
Illegal NgoMIV site found at 530
Illegal NgoMIV site found at 1032
Illegal NgoMIV site found at 1413
Illegal NgoMIV site found at 2338 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 577
Illegal SapI.rc site found at 660
Design Notes
cex normally contains domains GH10 (exoglucanase module) plus CBM2 (cellulose binding module). cenA contains a homologous CBM2 domain plus GH6 (endoglucanase module). It was therefore decided to fuse the two proteins at the CBM to make the new protein, designated cxnA, as follows:
- GH10 + CBM2 + GH6
By analysis of the matching CBM2 domains, it was determined that an NcoI site could easily be introduced into both at the same place without changing any amino acids.
BBa_K523016 (Plac + lacZα + RBS + cex) was amplified using forward primer pSBNX3insf2 (vector-based) and a new reverse primer which introduces an NcoI site in the CBM2 domain of cex.
cenA was amplified using a new forward primer which likewise introduces an NcoI site in the corresponding location of the CBM2 domain, and reverse primer pSBNX3insr2 (vector-based).
- primer cenAfNcoI: c ctg cca tgg aac ggc agc atc ccg acc
- primer cexrNcoI: c gtt cca tgg ggc gtt gcg gac cgt cac g
The two PCR products were purified, digested with NcoI and ligated, and the ligation was used as template for PCR with primers pSB1NX2insf2 and pSB1NX3insr2. Two major products were obtained. The larger, around 3.1 kb, was presumed to be correct, and was purified from the gel. The smaller, around 2.1 kb, was presumed to be a mixture of Plac-lacZα-cenA and Plac-lacZα-cex from intact template which had made it through the purification.
The purified insert was digested with EcoRI/PstI and ligated into pSB1C3. White colonies were tested for Cex activity on MUC plates. Positive clones were miniprepped and tested for insert size. Clones were further analysed by digestion with NcoI/PstI to check that the NcoI site was present. They were then tested for CenA activity on CMC plates.
It is therefore presumed, subject to confirmation of sequence, that clones which passed these tests represent the fusion of cex and cenA (i.e. Plac + lacZα + RBS + cxnA) and that this has both enzyme activities.
Source
Via PCR and other molecular techniques on extant parts.