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protein induction problem - (Feb/13/2011 )

Hello, guys. I recently have some problems to induce my proteins. I changed the lab and tried to induce same protein(same plasmid) in the new lab. But the induction decreased a lot. I checked the condition what I used in both labs, please help me to analyze which one is most possible reason.
1. IPTG from different companies
2. I used normal culture flasks in the new lab, but I used baffled culture flasks in the former lab.
3. I transferred the plasmid into BL21 gold DE3 in the new lab, and I used BL21 DE3 in the former lab.
4. incubator is different, but E.coli grows.

Thank you very much.
Cypress

-cypress-

cypress on Mon Feb 14 07:55:59 2011 said:


Hello, guys. I recently have some problems to induce my proteins. I changed the lab and tried to induce same protein(same plasmid) in the new lab. But the induction decreased a lot. I checked the condition what I used in both labs, please help me to analyze which one is most possible reason.
1. IPTG from different companies
2. I used normal culture flasks in the new lab, but I used baffled culture flasks in the former lab.
3. I transferred the plasmid into BL21 gold DE3 in the new lab, and I used BL21 DE3 in the former lab.
4. incubator is different, but E.coli grows.

Thank you very much.
Cypress

Hola, in both labs, have you checked constitutive expression, before induction?. Try to control it adding 0.2-0.5 g/l glucose , check expression before induction and look if this way recover your recombinant protein levels. Buena suerte

-protolder-

Hola, in both labs, have you checked constitutive expression, before induction?. Try to control it adding 0.2-0.5 g/l glucose , check expression before induction and look if this way recover your recombinant protein levels. Buena suerte



Thanks Buena,
Yes, protein had very little or no constitutive expression before induction. Could you tell me why do we need glucose,thank you.

-cypress-

Hola, The idea is control expression giving some repression. Whithout glucose the cAMP levels are high, these strains haven´nt any barrier for lactose (Natural inducer) as LaqIq or lacY minus (permease) and growing at 28ºC could lead to have your strain ready to be induced. Other possibility would be to check the expression in one of the BL21 lacIq or lac Y- strains. Buen día

-protolder-

protolder on Mon Feb 14 09:35:53 2011 said:


Hola, The idea is control expression giving some repression. Whithout glucose the cAMP levels are high, these strains haven´nt any barrier for lactose (Natural inducer) as LaqIq or lacY minus (permease) and growing at 28ºC could lead to have your strain ready to be induced. Other possibility would be to check the expression in one of the BL21 lacIq or lac Y- strains. Buen día


Hola,

I had induction problems as well.
I cannot utilize glucose since I'm developing a system alternative to it, and I'm using chemically defined medium, so no other carbon sources are present.

I reckon tight repression would be an issue only if the protein were extremely toxic for the host, otherwise a leaky promoter won't necessarily stop plasmid propagation.

My strategy for trouble shooting at the moment is to check whether my IPTG is viable, and luckily I have BL21(DE3) that works.
Secondly, I'll transform the 'difficult' plasmid into BL21 and MG1655.

However, regarding the original points:

1. try if IPTG works, include a positive control if you have it (i.e.: a vector that expresses). If you don't you could plate 'any' wild type strain with IPTG and Xgal;
2. baffled flasks kill your E. coli only if cellular membrane is weakened (i.e.: overexpression of membrane proteins);
3. the two BLs should be more or less the same (here, here and here), unless you protein has rare codons for E. coli;
4. not a worry.

I hope this helps a little and any other comment is welcome.
Sorry for the long post.

Suerte en todo.

M

-miche-