BIO 326R 1st Edition Lecture 19 Outline of Last 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 fusionOutline of Current Lecture I. reporter genesa. beta galactosidaseb. GFPc. Lux CDABEII. Triparental conjugationIII. Biparental conjugationCurrent LectureReporter Genes- Beta galactosidaseo lacZ= gene, LacZ=proteino cleaves lactose to allow growth on lactoseo ONPG= ortho-nitrophenyl- beta galactosidase When nitrophenyl is cleaved to glactose + o-nitrophenyl turns yellow- The more yellow, the more enzyme (beta galactosidase) present- GFP (green fluorescent protein)These 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.o Promoter-gfp=> measures green fluorescence Blue light (~488 nm) green light (510 nm)- Excite with blue, emits greeno Advantages over LacZ No substrate addition No cell lysiso Disadvantages over GFP Low sensitivity Narrow dynamic range—only a certain amount can be used- LuxCDABE—makes 5 proteinso Operon=> produces visible light= luminescence o Advantages Extremely sensitive: can count photonso Disadvantages Require massive amount of energy to make light- (10,000- 100,000 ATP/sec) Causes slow growth Resistant to antibioticsTriparental Conjugation- Three strain matingo Helper strain= self transmissible plasmid Plasmid with tra, mob, and oriTo Donor strain= has the plasmid you want to move to the acceptor Plasmid with mob and oriTo Acceptor strain= strain we want to introduce the plasmid into- Optimal if helper strain plasmid is a suicide plasmid for acceptor so if it goes straight to the acceptor, it won’t stay—we want it to go to the donor strain plasmid firstBiparental Conjugation- Self transmissible plasmid from donor to acceptoro Donor has tra, oriT, and
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