Difference between revisions of "Part:BBa K2940002"

Revision as of 02:33, 21 October 2019

Improved Bpul

Improved Bpul (bacterial laccase Bpul from Bacillus pumilus)

Usage and Biology

Laccases are enzymes with the potential to biodegrade synthetic dyes with different chemical structures. These enzymes are able to oxidize a wide range of phenolic substrates without the presence of additional co-factors. This improved Bpul enzyme has a major bioactivity for degrading azo dyes.

Characterization

Libraries of laccase mutants were generated using error prone Taq PCR (New England BioLabs). The mutant libraries of assembled PelB and Bpul were then cloned into pET28a-GG-RFP-CD via Golden Gate cloning for expression.

Characterization ABTS assay

98 mutant laccase enzymes were assayed for enzymatic activity using ABTS assay. This assay showed four mutants with improved activity (one with a significant increase). This mutant was sequenced and showed eight-point mutations when compared to WT Bpul.

Results

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 726
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 123
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 726
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 726
Illegal NgoMIV site found at 490
Illegal NgoMIV site found at 958
Illegal AgeI site found at 246
Illegal AgeI site found at 1123
1000
COMPATIBLE WITH RFC[1000]

@@ Line 28: / Line 28: @@
 <span class='h3bb'>Sequence and Features</span>
-<partinfo>BBa_K2940003 SequenceAndFeatures</partinfo>
+<partinfo>BBa_K2940002 SequenceAndFeatures</partinfo>
 <!-- Uncomment this to enable Functional Parameter display
 ===Functional Parameters===
-<partinfo>BBa_K2940003 parameters</partinfo>
+<partinfo>BBa_K2940002 parameters</partinfo>
 <!-- -->