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Puteri Q's Content

There have been 9 items by Puteri Q (Search limited from 21-September 18)


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#88762 E.coli suddenly death??

Posted by Puteri Q on 04 October 2010 - 05:51 PM in Microbiology

Oook I see..
Thank you...



#88679 E.coli suddenly death??

Posted by Puteri Q on 03 October 2010 - 09:41 PM in Microbiology

The maximum OD600 is (only) 0.3 (4 Hours incubation).

adding some Glucose? Ummm...but I use transforman E.coli BL21 (DE3) pLysS harbouring pET. Is it OK? I mean, is it ok if we add glucose to a system which is operate Lac operon?
Well, it could be tried.

But, how about the sudden death?? I guess there is any toxin in the waste water. maybe something like heavy metal...(I dont know). still on investigation.

:blink:



#88392 E.coli suddenly death??

Posted by Puteri Q on 29 September 2010 - 09:30 PM in Microbiology

Hi all..

I grew E.coli in certain wastewater that enriched with yeast extract. I measure the growth rate by OD600. The bacteria seems to grow 'quite' good for 4 H. But, after that (6H, 12 H....till 24 H) the growth phase was suddenly decline. I am confused why the stationary phase occur briefly? The growth curve looks abnormal.

Is there anyone who know the reason why E.coli could suddenly death?
is it possible the bacteria doesn't have stationary phase?



#85704 How to grow more E.coli?

Posted by Puteri Q on 02 September 2010 - 10:41 PM in Microbiology

I also have the same problem. My research goal is to know when exponential phase of my E.coli is occur. Because, I use non standard media to grow E.coli. It has low amount of protein or Nitrogen source, therefore I added 0,5% yeast extract into it (ust like in Luria Bertani) to optimize the E.coli growth.
But, the growth curve always shows different pattern if I do it in different time. First time I did it, OD600=0.6 could be reached in 4 H. But at other times, E.coli only reached OD600=0,3 in 4 H. It is half of the previous result.
I do really confuse with this.
Is there anyone who could explain why bacterial growth rate in the same media could change? :unsure:

Advice please....



#83930 when should I add IPTG?

Posted by Puteri Q on 17 August 2010 - 10:50 PM in Protein and Proteomics

Ok. Thank you HomeBrew ^_^



#83892 when should I add IPTG?

Posted by Puteri Q on 17 August 2010 - 06:17 PM in Protein and Proteomics

Hii Bioforumers...

I need your help to overcome my research problem.

For expression of the recombinant enzymes, the transformed E. coli BL21 cells were grown in 1 liter of LB medium at 37C containing 100 g of ampicillin per ml usually induced in mid-exponential phase (OD600: 0.5-0.6) with 1 mM IPTG.
If we use alternative medium other than Luria Bertani, does it mean that we have to find out the exact time of mid-exponential phase? or is it ok if we add IPTG into the culture just when the OD600 is between 0.5-0.6?

please help me..



#83369 appropriate time for induction (adding IPTG)

Posted by Puteri Q on 13 August 2010 - 12:01 AM in Protein and Proteomics

Hii Bioforumers...

I need your help to deal with my research problem. It is about recombinant gene expression.

Based on the paper, induction of genes in E. coli (BL21) growing in Luria Bertani usually performed after reaching OD600 between 0.5-0,6. It is said to be coincide with the exponensial phase.
If we use another growth medium for expression, then we induce the culture with IPTG, should we know what time the bacteria reaching exponensial phase? or is it enough for us to add IPTG into the culture just when OD600 between 0.5-0.6?

Please help me to overcome this problem.

-_-



#78796 protein overexpression

Posted by Puteri Q on 08 July 2010 - 09:33 PM in Protein Expression and Purification

Hi there...
Ive been doing my research about protein overexpression. Is there anyone who knows any factor that may affect to protein expression?
Does the enviromental conditon (ex. medium) may affect to the solubility of the recombinant protein?

P.S.: transforman==> E.coli BL21 DE3 Plyss

--Q--
B)


Yes, the environment does affect the solubility and expression of recombinant protein. This is why we spend so much money and time optimising growth condition. And it is also the reason why it is so difficult to scale large bio reactors.

Examples of factors that affect protein expression,
Media Carbon-nitrogen ratio.
Carbon source composition.
Nitrogen source composition/availability.
Temperature
Oxygen availability
Waste product build up
Cellular growth rate
pH

It is complicated and i only touched the periphery of this field when I was reading up factors that influence plasmid production and cell growth density.



Ooo..thanks for the answer. :) Do you have any suggestion for the reference that I could learn about those factors?



#77833 protein overexpression

Posted by Puteri Q on 01 July 2010 - 07:26 PM in Protein Expression and Purification

Hi there...
Ive been doing my research about protein overexpression. Is there anyone who knows any factor that may affect to protein expression?
Does the enviromental conditon (ex. medium) may affect to the solubility of the recombinant protein?

P.S.: transforman==> E.coli BL21 DE3 Plyss

--Q--
:lol:




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