BIO 326R 1st Edition Lecture 18 Outline of Last Lecture I. Transductiona. Generalized transductioni. Lytic bacteriophageb. Homologous recombinationc. Transposonsi. Inverted repeatsii. transposaseOutline of Current Lecture I. Transposonsa. Properties of a good transposonII. Transcriptional regulationa. Sigma factori. RpoSb. Transcription factorsi. Activators, repressors, and inducersIII. Studying promoter activitya. Reporter gene fusioni. Transcriptional reporterb. Translational fusionCurrent LectureTransposons—Properties of a good transposon- Random insertiono Have specificity—but we DON’T want that or else transposon will always go to the same spot of the genome- Antibiotic resistance geneo Or for example, mercury resistance- Transpose at high frequency - Self transmissibleThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Suicide plasmid = won’t replicate in the acceptor/host bacteria o Eventually gets degraded all that’s left is chromosomes with inserted transposons if also self-transmissible, random insertion, transpose at high frequency- Drawback: can’t study nonessential genes- Transposase= trans actingo Plasmid with IR, Ab^r, and transposase gene (mini transposons)Transcriptional Regulation- Way that bacteria regulate behaviors- Most genes are regulated, usually by environmental stimuli- Gene expression= transcription- Inactivate the gene encoding regulation- Targeted mutagenesiso Make plasmid with PCR (suicide plasmid, self transmissible plasmid)o Chromosome with gene a and b adjacent (ToxA) homologous recombinationgene a, antibiotic resistance gene (between a and b), gene b- Regulation factorso Sigma factor= sigma 70 (housekeeping) RpoS= sigma s (stationary phase sigma factor)o Transcription factors= proteins, transcriptional regulators Activators and repressors- Repressors= affinity changes depending on presence or absence ofamino acids to repress or not repress transcriptiono Ex: high argenine cell does not need to transcribe this amino acid, repressor binds operator to prevent RNA polymerase binding- Activator = affinity changes dependent on present or absence of amino acid to activate or not activate transcriptiono binding site at -35o ex: in absence of an amino acid (or decrease in concentration of an amino acid), cell needs it, activator is bound Operator= sequence that transcription factors bind toStudying promoter activity- Make reporter gene fusiono Transcriptional reportero lacZ=> encodes beta-galactosidase enzyme that cleaves ONPG into galactose and ortho-nitrophenol (yellow)- translational fuctiono SD comes from gene or interesto Tells you about the promoter and SD Tells you about transcription and
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