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Chapter 6

BIOL342 Chapter 6- readings.docx

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University of Waterloo
BIOL 342
Christine Dupont

Chapter 6- Readings Manipulation of Gene Expression in Prokaryotes - Remember that the main purpose of gene cloning is to get the expression of the cloned gene in a host organism - However, sometimes inserting a gene into a cloning vector doesn’t always result in the gene being expressed - For many biotech purposes we require high amounts of protein production There are many ways to manipulate gene expression: - Via promoter and terminator sequences (transcriptional control) - RBS strength - Translation efficiency  There is no single strategy to get the maximal gene expression of a cloned gene  The level of gene expression also often relies on the host organism GENE EXPRESSION FROM STRONG & REGULATABLE PROMOTERS A main requirement for gene expression is to have a strong and regulatable promoter - A strong promoter will have a high affinity for RNApol Being able to regulate a promoter will allow for control of transcription So.. - It might seem like it’s a good idea to express a cloned gene through a continuously activated and strong promoter HOWEVER.. - High levels of continuous expression of a cloned gene isn’t always great, “leakiness/toxicity” - Continuous expression may lead to high energy drain, “metabolic load” - Large quantities of normally benign proteins can become toxic The most commonly used “regulatable” promoters are from the Lac and Trp operons - The tac promoter is and artificial hybrid promoter that has the -10 region of the lac promoter and the -35 region of the trp promoter (mutated) - The pL promoter is from bacteriophage lambda  Each of these promoters will interact with repressors and inducers in order to control turning on and off of transcription Now thinking about growth mediums.. - If there is no lactose, the lac promoter will be turned off by the lac repressor protein, this prevents the lac operon from being transcribed - Adding lactose will induce the lac promoter thus transcription occurs The different levels of glucose, lactose, and cAMP in a growth medium will have different effects on the level of transcription with respect to the lac promoter - Transcription with the lac promoter is regulated by the binding of the CAP to the CAP box which is upstream of the promoter - When CAP binds to the CAP box it will increase the affinity of the promoter for RNApolymerase - This therefore increases the level of transcription CAP affinity to the CAP box is enhanced with cAMP cAMP levels are highest with low levels of glucose when lactose of IPTG (inducers) are present, there is no repressor bound to the operator  this means cAMP levels are high, meaning high level of transcription of the genes Talking about the TRP promoter now… - The trp promoter is strong and is negatively regulated, meaning that it is turned off by the trp repressor protein complexed with tryptophan, which binds to the operator and stops transcription - To turn on the trp promoter, you would remove tryptophan Repression of the trp promoter is
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