Heterologous expression of a Chitinase gene from Serratia marcescens Bizio strain LPC Biotech 1748 in Escherichia coli (Migula) castellani and Chalmers strain BL21-SI

Date

4-2006

Degree

Bachelor of Science in Biology

Major Course

Major in Cell Biology

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Neilyn P Ona-Villa

Abstract

A cloned full-length chitinase gene of size 2.1 kb from Serra& marcescens Bizio strain LPC BIOTECH 1748 harbored in a plasmid was introduced in Escherichia coli (Migula) Castellani and Chalmers 13121-S1 strain through transformation. Introduction of the said gene into the new host (E. coli) was proven successful after amplification of the gene by Polymerase Chain Reaction (PCR). The PCR products of the transformants, resolved in 1.2% agarose gcl, showed a single band with a size of 2.1 kb. Ileterologous protein expression of the chitinase gene was induced by the addition of 0.3 M NaCI to the transformed E. coil cultures. To verify the expression of the chitinase and determine its molecular weight (MW), crude protein extract was subjected to SDS-PAGE. Results showed a distinct 20 kDa band in the transformed BL21-SI extracts which was absent in the untransformed 1121-S1 cell extracts. Transformed cells, both with and without salt treatment, produced the 20 kDa protein band in the periplasmic space but chitinase production was significantly higher in the former. There was a suspected truncation of the chitinase since the obtained MW was much smaller than expected. By immunodot blotting technique which made use of barley chitinase as antibody, it was proven that chitinase was present in the transformed protein extracts. Plate assay showed, however, that the chitinase is unable to degrade chitin. The non-functionality is attributed to the protein being truncated.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

Thesis

Document Type

Thesis

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