A commonly used plasmid is the pGEX series, in which the gene to be expressed is...
A commonly used plasmid is the pGEX series, in which the gene to be expressed is placed directly under the control of a lac promoter. Could leaky expression be controlled through the use of BL21(DE3)pLysS cells? Why?
Homework Answers
Sometimes lac promoter can “leak,” but it can be fixed by BL21(DE3)pLysS
Principle
BL21(DE3)pLysS - BL21 that has the T7 RNA polymerase gene under the control of the lacUV5 promoter.
This arrangement is on a phage genome, called DE3.
DE3 is inserted into the chromosome of BL21 to make BL21(DE3).
pLysS is a plasmid that contains the T7 lysozyme gene (LysS).
The T7 lysozyme binds to T7 RNA polymerase causing inhibition until induction by the addition of IPTG.
When IPTG is added, the amount of T7 RNA polymerase increases and overcomes the inhibition by LysS.
Reason
Usually protein products ,prevents the medium of culture to reach its final density within the time period so there will be low level of expression even without addition of IPTG.
High protein expression is achieved by IPTG addition
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gene whose activity is relatively easy to detect--is commonly used
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circumstances. Recombinant DNA technology is used to attach such a
reporter gene to the promoter of interest, after which the
recombinant molecule is inserted by transformation or transfection
into the cell or host organism of choice. Green fluorescent protein
(GFP) from the jellyfish Aequorea victoria fluoresces green when
exposed to blue light. Assume that you wished...
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