Hi, I am currently using BL21(DE3)PLySs for protein expression.
In our lab, we are using selective antibiotics for both inoculation(small culture, a.k.a over night seed culture) and scale up(Large culture). But while I was searching for some information about protein expression, I don't need antibiotics for scale up culture, and seems like it's better even for the protein expression.
Is it true? Are you guys using antibiotics for only inoculation?
Dear Hyunsoo
generally i'm ever maintain antibiotics in all the phases from the pre-culture to the scale-up. because it guarantee that cells remain able to maintain the plasmid during the growth. If it is true that with out antibiotic the cells are less stressed and can growth better, on the other side if a large % of it lost the plasmid during duplication you can have problem in terms of expression.
Of course it also depends from what do you mean for large scale. If large scale is high volume of culture but with a low cell density media, eg LB where cells can growth up to OD 2-3 or you are using high cell density media or feed batch fermentation wich guarantee high OD, which mean also higher number of cell duplication and therefore higher probability to lost the plasmid.
In industry some times we try to avoid the antibiotic in the scale-up but it is due the fact that is antibiotic also in trace cannot be present in the final products.
In this context industries are working to develope sistems alternative to the antibiotics to maintain the plasmid into the cells.
Attached you can find an hold patent application for development of one of this new yistem where we reported also some data regarding plasmid mantainance and protein expression in absence of antibiotic.
If you are interested in you can find some more information about high cell density media suitable for protein production in shake flask on my blog
ProteoCool (https://proteocool.blogspot.com/)
ciao
Manuele
Dear Hyunsoo
generally i'm ever maintain antibiotics in all the phases from the pre-culture to the scale-up. because it guarantee that cells remain able to maintain the plasmid during the growth. If it is true that with out antibiotic the cells are less stressed and can growth better, on the other side if a large % of it lost the plasmid during duplication you can have problem in terms of expression.
Of course it also depends from what do you mean for large scale. If large scale is high volume of culture but with a low cell density media, eg LB where cells can growth up to OD 2-3 or you are using high cell density media or feed batch fermentation wich guarantee high OD, which mean also higher number of cell duplication and therefore higher probability to lost the plasmid.
In industry some times we try to avoid the antibiotic in the scale-up but it is due the fact that is antibiotic also in trace cannot be present in the final products.
In this context industries are working to develope sistems alternative to the antibiotics to maintain the plasmid into the cells.
Attached you can find an hold patent application for development of one of this new yistem where we reported also some data regarding plasmid mantainance and protein expression in absence of antibiotic.
If you are interested in you can find some more information about high cell density media suitable for protein production in shake flask on my blog
ProteoCool (https://proteocool.blogspot.com/)
ciao
Manuele
I have been using the same strain, I use all the antibiotics in the small volume culture, but after that, I only use one of them (the one to maintain my plasmid of interest), i do not use chloramphenicol to maintain the pLysS plasmid since my protein is not toxic. I guess using one of the antibiotics (the one needed for the plasmid) will allow you to be sure that your plasmid is still in the bacteria, therefore your protein is being produced normally.
Thank you for your kind answer guys! I will read what Manuel attached and also try Jorgaq's comment. My protein is not toxic too, but just in case, I will try for one flask, haha.
I used the same strain and always use antibiotic at all phases. Antibiotics dont affect expression level.
The reason we use antibitics for large cultures in the lab is because we are not the only ones using the equipment so we're basically avoiding contamination. But when it comes to the expession procces we prefer to use only one antibiotic, the one we use to select the bacteria with the expression vector.
HI, Hyunsoo Cho
I do agree wth others and would keep up, at least, with the one used for selection purposes.
If you're new in the field and yet did not have a huge background on protein expression and purification and did not get any good resources, please, use this one as your basic material.
https://www.qiagen.com/us/resources/download.aspx?id=79ca2f7d-42fe-4d62-8676-4cfa948c9435&lang=en (It's free to be downloaded at the company's website).
I wish you all the best.
Actually, the purpose of using antibiotics is to avoid the contamination. So, you can use less amount of antibiotics and can check.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression