Arrange the steps of obtaining recombinant insulin protein in the correct order • Mix digested insert and plasmid • Add DNA ligase • Reverse transcribe human insulin mRNA • Mix recombinant plasmid and bacteria • Harvest human insulin protein • Cut plasmid and insert with restriction enzyme • Grow recombinant bacteria in liquid culture • Grow recombinant bacteria on ampicillinAnother important technique in biotechnology: Polymerase Chain Reaction (PCR) • Allows to obtain millions of copies of a DNA of interest, starting with a few (or a single) copy • Relies on thermostable DNA Polymerase • Requires 2 primers, specific to the ends of the sequence you want to amplify • Animation: http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swfFig.%20(8%5ʹ′"Genomic"DNA"TECHNIQUE"Cycle"1"yields"2"molecules"Denatura>on"Annealing"Extension"Cycle"2"yields"4"molecules"Cycle"3"yields"8"molecules;"2"molecules"(in"white"boxes)"match"target"sequence"Target"sequence"Primers"New"nucleoL">des"3ʹ′"3ʹ′"3ʹ′"3ʹ′"5ʹ′"5ʹ′"5ʹ′"1"2"3"Steps"in"PCR"reac>on"Regulation of Gene Expression in Prokaryotes and Eukaryotes • Chapter 18 • Animations: http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html#Why does a prokaryote cell need to regulate the expression of its genes? 1. In response to environmental changes 2. In response to its own metabolic needs Both in prokaryotes and in eukaryotes, the most important level of the regulation of gene expression is regulation of transcriptionRegulation of transcription is accomplished by transcription factors and regulatory regions Transcrip1on%factors%are%proteins%that%bind%to%the%regulatory%regions%of%the%gene%and%increase%or%decrease%the%rate%of%transcrip1on%of%the%gene.%The%regulatory%regions%are%o<en%called%operators%in%prokaryotes,%and%enhancers%in%eukaryotes.!%Transcrip1on%factors%that%ac1vate%transcrip1on%are%called%ac>vators.%Transcrip1on%factors%that%decrease%transcrip1on%are%called%repressors.%A sketch of a transcription factor MyoD and its DNA binding site The%α helices%are%represented%as%cylinders%Prokaryotic transcription regulation: tryptophan operon Operon:%a%set%of%genes%transcribed%into%one%mRNA.%Operons%are%a%unique%prokaryo1c%feature.Usually,%all%genes%in%the%operon%encode%for%pro teins%that%par1cipate%in%the%same%process,%such%as%the%five%enzymes%involved%in%tryptophan%synthesis.%How do repressors down-regulate gene transcription? Regulation of tryptophan operon Animation: http://highered.mcgraw-hill.com/olc/dl/120080/bio26.swfBacterial activator CAP interacts with RNA Polymerase and helps it initiate transcription efficiently CAP%has%to%bind%cyclic%AMP%(cAMP,%an%intracellular%signal%that%indicates%that%glucose%is%not%available)%in%order%to%be%able%to%bind%to%DNA%http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html# Lactose"(lac)"operon:"combina>on"of"posi>ve"and"nega>ve"transcrip>on"switches""Combina>on"of"posi>ve"and"nega>ve"regula>on"ensures"that"lactose"operon"will"be"transcribed"only"when"glucose"levels"are"low"and"lactose"levels" are"high""When"glucose"is"present"A) cyclic%AMP%is%high,%the%catabolite%ac1vator%protein%(CAP)%binds%to%the%ac1vator%binding%site,%and%transcrip1on%of%lactose%is%turned%on%B)%cyclic%AMP%is%low,%CAP%binds%to%the%site%ac1vato r%bind in
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