Engineering Success

Overview

Our experiment mainly consists of four parts.

1)Codon optimization

2)Vector construction

3)Competent Corynebacterium glutamate production

4)Inducible expression

Codon optimization

The initial length of bpsA gene was 3849bp, and was synthesized into two parts, which were 1900bp and 1970bp separately. A Flag-tag was added before the stop codon.

Vector construction

1.Amplification of two bpsA fragments. DNA Marker: 2000bp

2.Recombinant vector was transformed into DH5α. DNA Marker: 5000bp

3.Recombinant vector was transformed into Corynebacterium glutamicum. DNA Marker: 5000bp

Competent Corynebacterium glutamate production

Using Bio-rad power station instrument to perform the following electrical transfer steps:

1)Add 5 uL plasmid into competent Corynebacterium glutamicum cells at 1.8 kV/mm for 5ms;

2)Immediately add 915 uL of preheated LBHIS Buffer, at 46 ℃, and incubate for 6 min;

3)resuscitation at 200 RPM for 2 hours at 30 ℃;

4)Spread on Kana resistant plates and produce transformants in 1-2 days.

Inducible expression

The C.glu-bpsA strain was directly streaked on LBG(K+) solid plate with IPTG at final concentration of 1mM, and cultured in incubator at 37℃ for 1-2 days.