Difference between revisions of "Part:BBa K2302005"

Revision as of 10:39, 27 October 2017

pelB,a signal peptide that helps the transportation of a gene product out of the membrane.

This sequence encodes a functional signal peptide that can promote the transportation of its fused protein out of the membrane.

Design

To express and secret recombinant CAlB in E. coli, the 5’ terminal of the CALB gene without its own translational initiation codon was combined with the genes coding for the original Pelb signal sequence in Pelb-CALB#1, and Pelb-CALB#2 was combined with improved Pelb signal sequence. The gene of fusion protein was constructed in the pHis vector with tac promoter.

Experiment and Result

To further demonstrate whether the CALB can secrete to the medium by the signal peptide Pelb, the supernatant was directly detected using western blot. CAlB was targeted by His-tag probe. 30ug of protein sample was added in every well. It was obvious that the bands were much more darker in figure B, which indicated the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1. The predicted molecular weight of fusion protein is different between Pelb-CALB#1 and Pelb-CALB#2, it maybe caused by the changed protein conformation after the codon optimization. Comparing the expression between A and B, it shows that the function of Pelb has been improved remarkablely.

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 25
Illegal NgoMIV site found at 51
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 This sequence encodes a functional signal peptide that can promote the transportation of its fused protein out of the membrane.
-<!-- Add more about the biology of this part here
+===Design===
-===Usage and Biology===
+To express and secret recombinant CAlB in E. coli, the 5’ terminal of the CALB gene without its own translational initiation codon was combined with the genes coding for the original Pelb signal sequence in Pelb-CALB#1, and Pelb-CALB#2 was combined with improved Pelb signal sequence. The gene of fusion protein was constructed in the pHis vector with tac promoter.
+===Experiment and Result===
+To further demonstrate whether the CALB can secrete to the medium by the signal peptide Pelb, the supernatant was directly detected using western blot. CAlB was targeted by His-tag probe. 30ug of protein sample was added in every well. It was obvious that the bands were much more darker in figure B, which indicated the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1.
+The predicted molecular weight of fusion protein is different between Pelb-CALB#1 and Pelb-CALB#2, it maybe caused by the changed protein conformation after the codon optimization. Comparing the expression between A and B, it shows that the function of Pelb has been improved remarkablely.
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