Team:Lethbridge/ACK Production

Results

Overexpression of ACK (BBa_K901015)

Overview

To test whether the construct for the acetogenesis protein ACK was working as expected, we tested the overexpression of ACK in E. coli after induction by IPTG.

Experimental Setup

Overnight cultures of E. coli were used to inoculate fresh LB media to a starting optical density (OD) of 0.1. At an OD600 of 0.6, the expression of the constructs was induced by the addition of 1 mM IPTG. After induction, 1 OD600 of cells were taken at 0.5, 1, 2, and 3 h for analysis by SDS-PAGE.

Results

The expected size of ACK is 43 kDa. The samples from the overexpression were analyzed by 12% SDS-PAGE (Fig. 1). When compared to the protein molecular weight marker, there is a band that appears around the 45 kDa mark. The protein EF-Tu is of a similar size and could be contributing to the band seen at this size. However, there appears to be an increase in intensity when comparing the sample from 0 h after induction to 3 h after induction. This suggests that ACK was overexpressed and the expression of the construct is working as expected.

Figure 1. 12% SDS-PAGE analysis of overexpression of ACK by E. coli. Lane numbers indicate time in hours after induction with IPTG; L indicates ladder. The black arrow indicates the band showing overexpression of ACK.