Experimental Procedure
Bacteria were grown in 37oc to OD600 of 0.6. At this point the RT (room temperature) flasks were transferred to 25oc and allowed to accomodate to the new tempearture for 15 minutes prior to induction. IPTG (0.2; 0.5 or 1.0 mM) was than added. Cultures were harvested 2, 4 and 16 hrs post-induction. Cells were lysed, sonicated and centrifuged (14,000 rpm, 15 minutes, 4oc). Samples of Supernatant and Pellet were run in PAGE-SDS gels and subjected to Commassie staining or western blot analysis using anti-GST antibodies. All western blot images were obtained following an exposure of 10 seconds.
Note: the first two samples on the left of each figure (1.0 P and S) are samples of the control GST-GFP system (expected size ~55 KDa) grown in 37oc and induced by 1mM IPTG.

Scroll down to see ALL results.

Conditions
Coomassie Staining
Western Blot (anti-GST ABs)
OrigamiB pLysS 2 hrs
OrigamiB pLysS 4 hrs
OrigamiB pLysS overnight
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Q3
Go through the following observations and choose "True" or "False" for each of them.
There may be more than one "right" answer.
1. The Commassie results at all time points show that GST-GFP (~55KDa) is produced, and no problems are observed.

2. The produced GST-GFP protein is insoluble, as evident by Commassie staining and western blots.

3. The Commassie results at all time points don't reveal differences between the expression temperatures (37oc and 25oc) and inducer's concentrations (0.2, 0.5 and 1.0 mM).

4. The Commassie results of all the NS4B expression samples show two main specific products (fragments ~35 and 40 KDA), which are insoluble and somewhat shorter than the expected product.

5.  Western blot analysis of 4 hrs post-induction samples indicates that samples grown in 37oc give higher protein yields than samples grown in 25oc.

6. Western blot analysis of all time points does not reveal major differences between different inducer concentrations.

Have you examined all the answers?
Continue.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Conclusions

These results indicate that Origami cells support very low levels of NS4B synthesis (observed only by western blot analysis). The best growth conditions are 25oc, 4 hrs of induction using 0.2 or 0.5 mM IPTG.  Even under those conditions the protein is insoluble and some of the produced protein is of smaller size than expected.

Let's examine the growth curves of this strain under the various growth conditions.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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