Difference between revisions of "Part:BBa K1350007"

Latest revision as of 07:02, 17 October 2014

Cel5A, endoglucanase（BBa_K805011 Improved）

The endoglucanase genes cel5A can hydrolyze β-1,4glucosan randomly and act on long cellulose chains. The main product is oligomeric glucose. It can digest glucosan to produce glucose monomer in high efficiency with synergy with β-1,4glucosidase.It shows high ability to hydrolyze the crystal cellulose but relative lower enzyme activity towards carboxymethyl cellulose, barley β-glucan, and PASC.

Improved endoglucanase Cel5A

BBa_K805011[1] given by iGEM12_HUST-China had BamH I site. The site is cleared by introducing a silent mutation. GGATCC (874-879) has been changed to GGGTCC. A codon GGA(Gly) is changed to GGG(Gly). Because of the degeneracy of codon,the different sequence can encode the same enzyme.This silent mutation makes the part can be compatible with RFC[21].

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 NgoMIV site found at 307
Illegal NgoMIV site found at 763
Illegal AgeI site found at 1027
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1024

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1350007 short</partinfo>
-we got this endoglucanase from iGEM kit,and we mutate it in a certain spot.[https://parts.igem.org/Part:BBa_K805011]<br>
-===Cel5A, endoglucanases===
 The endoglucanase genes cel5A can hydrolyze β-1,4glucosan randomly and act on long cellulose chains. The main product is oligomeric glucose. It can digest glucosan to produce glucose monomer in high efficiency with synergy with β-1,4glucosidase.It shows high ability to hydrolyze the crystal cellulose but relative lower enzyme activity towards carboxymethyl cellulose, barley β-glucan, and PASC.
-===Usage and Biology===
+===Improved endoglucanase Cel5A===
-The expression levels of cel5A is repressed by glucose and induced by cellulose.It can hydrolyze β-1,4glucosan randomly and act on long cellulose chains. The main product is oligomeric glucose. It can digest glucosan to produce glucose monomer in high efficiency with synergy with β-1,4glucosidase.It shows high ability to hydrolyze the crystal cellulose but relative lower enzyme activity towards carboxymethyl cellulose, barley β-glucan, and PASC.
+BBa_K805011[https://parts.igem.org/Part:BBa_K805011] given by iGEM12_HUST-China had BamH I site. The site is cleared by introducing a silent mutation. GGATCC (874-879) has been changed to GGGTCC. A codon GGA(Gly) is changed to GGG(Gly). Because of the degeneracy of codon,the different sequence can encode the same enzyme.This  silent mutation makes the part can be compatible with RFC[21].
-We construct the gene on the carrier displaying on the surface of Pichia pastoris and express it it in Pichia pastoris with electroporation. Then we detect the expression on the surface of Pichia pastoris by observing with fluorescent microscope and by flow cytometry analysis.
-[[Image:Luorescent_microscope_3.jpg‎]]
-We fused FLAG-tag with N-terminal of cel5A gene on cel5A-pPIC9k vector. Label cells with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody. Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPIC9k empty vector is 1.14, with cel5A-pPIC9k is 5.55.
-[[Image:Hust_part3.jpg]]
-[[Image:Hust_part4.jpg]]
-Left: pPIC9k negative control (x-mean: 1.14/13.4); Right: cel5A (x-mean: 5.55/16.5)
-<!-- Add more about the biology of this part here
-===Usage and Biology===
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1350007 SequenceAndFeatures</partinfo>
 <!-- Uncomment this to enable Functional Parameter display