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Study Guide 2 BIO 463

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BIO 463

Why protect nitrogenous bases Bases adeinin, guainie and cyosine are derivatized by the addition of benzyoyl, isobutyrl and benzyol groups, respectively to prevent undesirable side reactions during the chain growth. Thyimine is nto treated because it lacks amino groups. Chp 6: 1. How a gene can be regulated by Pl promoter? a. The ability to regulate a promoter enables the cell to control the extent of transcription in a precise manner. PL promoter controlled by CI repressor protein of bacteriophage. A temperature sensitive mutant of the cI repressor generally used to regulate pLdirected transcription. Cells carrying the cI repressor prevents transcription directed by the pL promoter. When cell culture reaches to desired stage of growth, temperature shifts to 42C so thermosensitive cI repressor is inactivated and transcription can proceed p.199 2. What is the role of cI gene in this regulation? a. The cI repressor protein controls the pL promoter. This is a temperature sensitive mutant and used to regulate pL directed transcription. Temperature sensitive and binds to PL promoter. If temperature is increased, than the cI will become inactive and he pLwill begin transcription an produce the plasmid’s copy number. 3. How can you control the expression of a gene using PT7 promoter? a. You can control the gene expression using pT7 promoter by adding IPTG. Without inducer IPTG, the lac repressor represses the synthesis of the T7 RNApolymerase that is transcriptionally controlled by the lac operator (oLac) and lac promoter (pLac). The lacI gene produces the lac repressor. When lactose or IPTG is added to the medium, it binds to the lac repressor preventing it from repressing the transcription of T7 RNApolymerase. The T7 RNA polymerase will transcribe the target gene. 4. What is a fusion protein? Why synthesized? How it is created? a. Target protein that is in frame with a stable host protein. Combined single protein protects the cloned gene product from attack by host cell protease. b. Resistant to degradation when they are part of fusion protein 5. How can it be separated? Proteolytic enzymes (proteolysis) p, 207 A. Xa linker- used to release target protein from the fusion partner because factor Xa is a specific protease that cleaves peptide bonds uniquely on the C-terminal side. B. Marker peptide –Antibody column purification Secreted fusion protein can be purified in a single step by immunoafinity chromatography. Monoclonal antibodies against the marker peptide have been immobilized on the polypropylene support and act as ligands to bind the fusion protein. C. His tag- protein and purification- Six or eight histidine residues attached to either N or C terminal end of the target protein. The histidine tagged protein alone with other cellular proteins is then passed over an affinity column of nickel-nitrioltracetic acid. The histidine tagged protein, but not the other cellular proteins binds tightly to the column. The bound protein is eventually eluted from the column by the addition of imizole (the side chain of histidine). D. intein tagged and purification- If proteases cleave POI at unintended sites and left a portion of the POI attached to its fusion partner, than the cleavage reaction may not go to completion. Intein can be used to catalyze its own cleavage into two separate polypeptides. (6.15) 6. Large scale preparation: a. Why use dual plasmid? To use for large-scale fermentations. Two plasmid system has been developed. The cI repressor was placed under the control of the trp promoter. The cI repressor was placed under the control of the trp promoter and inserted into a low copy number plasmid. The use of a low copy number plasmid ensures that excess cI repressor molecules are not produced. The second plasmid carries a cloned gene under the control of the pL promoter. In Fig 6.8a, the trp promoter is turned on in the absence of tryptophan, so the CI repressor protein is synthesized and the pL promoter is turned off. In contrast, in 6.8B, the trp promoter is turned off in the presence of trypophan so the cI repressor protein is not synthesized and the pL promoter is fully active. 7. Usefulness of inducible promoter? a. The ability to regulate a promoter enables the cell to control the extent of transcription in a precise manner. Each of these promoters interacts with regulatory proteins which provide a controllable switch for either turning on or turning off specific transcription of adjacent cloned genes. 8. Regulation of gene expression using a. Trp- Trp promoter regulates transcription of the genes that are necessary for the biosynthesis of the amino acid tryptophan. This strong pr
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