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Week 11 - LacZ fusions, PCR lig

Week 11 - LacZ fusions, PCR lig - BIOL 519 Week 11 LacZ...

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BIOL 519 Week # 11- LacZ fusions- PCR and Ligations 1 BACKGROUND This week we will begin a series of experiments that we will refer to as “LacZ fusions”, and that will continue for 3 weeks. The overall goal of this experiment is to construct a translational fusion between the lacZ gene and the promoter region of the rhamnose operon ( rhaBAD ), and then to quantify expression from the fusion in the presence and absence of rhamnose. LacZ fusions - A powerful tool in the study of gene regulation is the construction of transcriptional or translational fusions between the regulatory elements (promoter, etc.) of a gene of interest and the lacZ reporter gene. The lacZ gene is very useful as a reporter gene since a variety of substrates have been developed that can be cleaved by the LacZ enzyme to produce a colored product, allowing easy detection of lacZ expression both on plates and in quantitative assays. The two substrates we will use in these experiments are X-gal and ONPG. We discussed X-gal last week and similar to last week, we will continue to use X-gal to detect LacZ enzyme activity within colonies on plates. ONPG stands for o-nitrophenyl- β - D - galactoside. Prior to cleavage by LacZ, ONPG is colorless, however after cleavage one of the products of ONPG cleavage is yellow in color. ONPG is generally used as the substrate in quantitative assays of LacZ expression in liquid cultures. The amount of yellow color produced can be accurately measured using a spectrophotometer. In addition to the variety of useful substrates that are available to detect LacZ activity, there is a second reason why it is often simpler to assay LacZ expression rather than directly assaying the gene of interest. In contrast to the LacZ enzyme, detecting and assaying expression of the gene product normally produced from the gene of interest may be difficult, dangerous (if the assay requires acids or radioactivity, for example), or extremely expensive (if the assay requires a substrate that is unusual). Further, the expression from a variety of different promoters can be directly compared if all the promoters are fused with lacZ . Transcriptional fusions - In a transcriptional fusion, the lacZ gene is placed downstream of the promoter region of a gene or operon of interest. The lacZ gene provides its own translational signals (ribosome binding site = Shine-Delgarno + ATG) but depends on the transcriptional signals (promoter, activators, repressors, etc.) of the gene of interest. This kind of fusion allows one to quantify the strength of the promoter, as well as to determine the conditions that increase or decrease transcription from the promoter. For example, if geneX is normally only transcribed when the substrate “X” is available to the cell, then expression of a geneX-lacZ fusion would be turned off when the cells are grown in the absence of substrate X, and turned on when the cells are grown in the presence of substrate X.
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