OD as a function of time

13  - control , 37°C, 0.2 mM

14  - control , 37°C, 0.5 mM

15  - control , 37°C, 1.0 mM

16  - control , 25°C, 0.2 mM

17  - control , 25°C, 0.5 mM

18  - control , 25°C, 1.0 mM

19  - GST-NS4B, 37°C, 0.2 mM

20  - GST-NS4B, 37°C, 0.5 mM

21  - GST-NS4B, 37°C, 1.0 mM

22- GST-NS4B, 25°C, 0.2 mM

23- GST-NS4B, 25°C, 0.5 mM

24- GST-NS4B, 25°C, 1.0 mM
Note: the spectrophotometer's measurements are linear only up to 1 OD. Thus, samples measured at  various time points post-induction should be diluted. This was not done in this experiment. Luckily, the differences between the various experimental conditions are so marked, they can still be noted.
The growth of three samples (19, 20 and 21 = GST-NS4B , 37°C) is fully arrested at time intervals longer than 2 hours after induction. (These measurements are accurate since they fall below 1 OD.) Three additional samples (22, 23 and 24 =  GST-NS4B, 25°C) grow somewhat better, but function less well than the control samples at 4 and 16 hours post-induction. [Had the measurements been taken in the linear range, even greater differences would have been observed.]

Induction of NS4B synthesis has toxic effects - it arrests bacterial cell growth.
At 37oc protein synthesis progresses faster, and thus the growth arrest effects are more profound.

Apparently, very low amounts of NS4B were synthesized (as evident by the Commassiie staining and western blot analysis), perhaps because cell growth was arrested very soon after induction. NS4B's involvement in transcription and its ability to downregulate protein synthesis (which we learned about during our initial data accumulation) may account for such growth arrest.
Continue.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Q4
How can we improve NS4B expression?
Read ALL answers and choose between "right" and "wrong".
There may be more than one "right" answer.

1. Try another bacterial strain, which gives a higher protein yield such as BL21(DE3).

2. Grow another bacterial strain to an OD of 2.0, than switch to 25oc and induce synthesis for short time intervals (30-60 minutes) by adding 0.2mM IPTG.

3. Use a richer growth medium to support better yields of protein synthesis.

4. Attach a secretion signal to the N-teminus of NS4B. NS4B in the periplasm may prove less toxic to culture growth.

5. An original idea - abandon the project !!!

Have you examined all possibilities?
Let's examine the results obtained using the Rosetta bacterial strain.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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