Biotechnology from bench to business

• Follow GEN on:
• Login | Register | Subscribe

Optimizing Expression in an E.coli System

Thermo Scientific MaxQ 8000 Shakers Allow All Flasks in an Experiment to Be Run Simultaneously

• Mark Schofield

(Page 3 of 3)

Results

Figure 2. Half-normal plot showing statistical relevance of the three experimental factors Click Image To Enlarge +

Figure 2. Half-normal plot showing statistical relevance of the three experimental factors

Using the Design-Expert 7 Workstation, all three factors (temperature, TOI, and media) were shown to have statistically important effects on the level of soluble protein X expression, as judged from the half-normal plot Figure 2. Half-normal plots compare the absolute values of ordered residuals from the data, to the expected values of ordered observations from a normal distribution to establish which experimental effects are important and which are unimportant.

Discussion

Figure 3. Plot of the effects of temperature, time of induction (TOI) using either Super Broth (A) or LB (B) Click Image To Enlarge +

Figure 3. Plot of the effects of temperature, time of induction (TOI) using either Super Broth (A) or LB (B)

The procedure outlined in this article describes a method for the optimization of soluble protein expression in E. coli using the MaxQ 8000 refrigerated stackable shakers. Using the shakers we were able to grow cultures at different temperatures and study the effect of temperature, time, and media on the amount of soluble protein X produced. All three factors were seen to have statistically important effects on the level of soluble Protein X expression as judged from the half-normal plot. Interestingly, the effect of increasing both temperature and TOI was different for the two media. This experiment not only shows the importance of optimizing incubation parameters for recombinant protein expression in E. coli, but also that it can be achieved over hours rather than days when using good experimental design, coupled with superior equipment.

Key to the success of this investigation was the ability to design the experiment to run all flasks simultaneously using only two separate, stackable refrigerated shakers—the Thermo Scientific MaxQ 8000.

The MaxQ 8000 shaker range includes both refrigerated and incubated models and can be stacked (up to three units high), providing optimal use of any available floor space. In addition, the slide-out platform provides easy and rapid access to all the samples, during loading, unloading, and induction, for example. As a result, multiparameter, multilevel studies, such as the one demonstrated here, can be conducted very efficiently.