Team:Lethbridge/InvA and GalU

Results

InvA and GalU (BBa_K901003 & BBa_K901007)

Overview

To test whether the constructs for enhanced glucose production are working as expected, we tested the overexpression of InvA and GalU in E. coli as induced 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 InvA and GalU are 59 kDa and 33 kDa, respectively. The overexpression samples were analyzed by 12% SDS-PAGE. Figure 1 shows the overexpression gel for InvA. A slight band just below the 66.2 kDa molecular weight marker appears to become more intense as time after induction increases from 0 to 3 h. This indicates that InvA has been overexpressed, although expression levels are not very high. To increase expression levels, we can try to modulate the amount of IPTG added for induction, grow the cultures for a longer period of time, or try different incubation temperatures to facilitate expression of the protein.

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

Figure 2. 12% SDS-PAGE analysis of overexpression of GalU by E. coli. Lane numbers indicate time in hours after induction; L indicates ladder.

Figure 2 shows the samples from the overexpression of GalU. We were expecting to see a band of increasing intensity at around 33 kDa, however there are no bands that appear to increase in intensity. Overexpression of GalU was therefore not successful. To achieve overexpression, we will test conditions similar to those increasing expression of InvA.