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How OD600 influence on IPTG induction and protein expression

OD600 induction

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#1 Rchqing

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Posted 24 August 2012 - 09:02 PM

I want to know how OD600 influence on IPTG induction and protein expression? Take for example. 100ml overnight culture is transfered to 1L fresh media and the start OD600 is 0.4. If it was induced when OD reaches 0.6, would it be OK? Thank you!

#2 Inbox

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Posted 24 August 2012 - 10:07 PM

Bacteria follows the Lag, Log, stationary and decline phase of growth in a limited amount of medium. If you have to take bacteria in log phase of growth so it express good. More overnight culture you take means you are running risk of taking O.D. for Stationary / Decline phase culture (overnight O.D.) than from log phase.

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Edited by prabhubct, 24 August 2012 - 10:09 PM.


#3 Rchqing

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Posted 25 August 2012 - 12:00 AM

Bacteria follows the Lag, Log, stationary and decline phase of growth in a limited amount of medium. If you have to take bacteria in log phase of growth so it express good. More overnight culture you take means you are running risk of taking O.D. for Stationary / Decline phase culture (overnight O.D.) than from log phase.

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Do you mean the start OD of 0.4 is too large if I want to induce at OD=0.6? What is the start OD do you usually use for protein expression? Thank you!

#4 Rchqing

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Posted 25 August 2012 - 12:28 AM

Why people always induce in log phase? Is it because bacteria's physiological status in this stage is most suitable for induction, or because there is enough nutrients for expression, or some other reasons?

#5 ascacioc

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Posted 25 August 2012 - 11:03 AM

In different phases bacteria have a different internal machinery determined by different expression levels of different essential proteins. In the log phase the internal machinery of the bacteria is set to produce lots of proteins and fast division since enough food is available. For proper expression you need to have enough cell mass to produce your protein and these bacteria need to have enough healthy transcription/translation machinery to be able to produce your protein. This is ideal in the fast growing stage.

However, when your protein is toxic to the cells (after induction at OD600 of 0.6, the cells stay at ~0.6 for many hours), you might want to consider induction at 1.2. Moreover, there are people who induce at 0.3 (beginning of the log phase).

Andreea

#6 Rchqing

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Posted 30 August 2012 - 03:28 AM

In different phases bacteria have a different internal machinery determined by different expression levels of different essential proteins. In the log phase the internal machinery of the bacteria is set to produce lots of proteins and fast division since enough food is available. For proper expression you need to have enough cell mass to produce your protein and these bacteria need to have enough healthy transcription/translation machinery to be able to produce your protein. This is ideal in the fast growing stage.

However, when your protein is toxic to the cells (after induction at OD600 of 0.6, the cells stay at ~0.6 for many hours), you might want to consider induction at 1.2. Moreover, there are people who induce at 0.3 (beginning of the log phase).

Andreea

Thank you very much!





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