Q1: What underlying mechanism ensures that the Dual Media Set? works so reliably for protein overexpression?
Our research has found that the use of the common Luria Broth (LB) medium produces relatively high background levels of recombinant protein expression even without the inducer (such as IPTG). The expression often does not increase enough after addition of the inducer. We have concluded that although LB is an excellent medium for normal?E. coli manipulation, it is not a good medium for repression of protein expression during cell expansion, nor is it ideal for protein expression when inducer is added. EB? and OB? were designed to overcome these problems. EB? was designed to repress recombinant protein expression by regulating the levels of cyclic AMP (cAMP) and cAMP receptor protein (CRP) during cell expansion, so that the cells can be expanded without pressure of undesired foreign protein expression and unintentional selection of mutated clones to overgrow original inoculates. When the cells are expanded, OB? medium is added for protein expression. During this process, further cell growth is not required and selection of new mutants no longer occurs because cell replication is very limited at this stage. The carefully formulated composition of OB? works similarly by regulating the levels of cAMP and CRP but with an opposite effect than EB?. Therefore, addition of IPTG is not needed for strong promoters such as T7.
Q2: What are the ingredients of EB? and OB? ?
Our growth media contain only standard commonly used ingredients, salts, and carbohydrates. The final composition, and the ratio of individual components, has been carefully optimized to result in above described metabolic effects.
Q3: Can I replace EB? or OB? with my home made media?
EB? can be replaced with LB when the expressed protein is stable and does not interfere with cell growth. OB? cannot be replaced.
Q4: Can I use OB? without the initial cell expansion in EB? ?
The simplified protocol can be used, but only for most stable and easily expressed proteins. You can simply start at step 2. in Protocol I. This will result in strong protein overexpression, with the risk of introducing unwanted mutations or other unwanted effects.
Q5: I have always used IPTG to induce protein expression in the T7 system, why not now? Will I get less protein if I don?t use IPTG in such case?
The T7 promoter is too strong when induced with IPTG. The metabolic effects caused by growth in OB? are also strong. This is a powerful combination, always resulting in too rapid protein expression with negative effects on cell viability. This situation results in lower yields of recombinant protein and may lead to additional plasmid/protein instability.
|M3012-100||Expansion Broth (EB)||100 ml|
|M3013-100||Overexpression Broth (OB)||100 ml|