Figure 24.3: T7 Promoter mediated protein production control mechanism in expression host.

Expression of gene using E.coli expression system- The steps in an expression of a gene is outlined in Figure 24.4 and it has following steps:

1.Transformation- As discussed we can use multiple methods to transform the host with a recombinant clone containing suitable selection marker.

2. Growth of the bacteria- A single colony of the transformed colony is inoculated in the suitable media as discussed before upto the log phase (OD=0.6-0.7).

3. Induction- The bacterial culture is now induced with IPTG (0-1mM) for 3-6 hrs to produce the protein.

4. Recovery of the bacteria and analysis of protein expression- Bacteria can be recovered from the culture with a brief centrifugation at 8000-9000 RPM and analyzed on a SDS-PAGE. The details of SDS-PAGE will be discussed in a future lecture. The SDS-PAGE analysis of a particular expression study in E.coli is given in Figure 24.4 and it indicates a prominent expression of the target protein in the induced cells as compare to the uninduced cells.