1. Sadia Sayed

2. The expression of the cloned gene in a selected host organism. It does not necessarily ensure that it will be successfully expressed. A high rate of production of the protein encoded by the cloned gene is required. Specialized expression vectors have been created that provide genetic elements for controlling transcription, translation, protein stability, and secretion of the product from the host cell.

3. The promoter and transcription terminator sequences. The strength of the ribosome-binding site. The number of copies of the cloned gene. The gene is plasmid-borne or integrated into the genome of the host cell. The final cellular location of the synthesized foreign protein. The efficiency of translation in the host organism. The intrinsic stability within the host cell of the protein encoded by the cloned gene.

4. The strong promoter is one that has high affinity for RNA polymerase that the adjacent downstream region is frequently transcribed. The ability to regulate a promoter enables the cell to control the extent of transcription in a precise manner. The lac operon of E. coli has been used extensively for expressing cloned genes. Many different promoters with distinctive properties have been isolated from a range of organisms.

5. The most widely used are those from the E. coli lac and trp operons. The tac promoter is constructed from -10 region of the lac promoter and -35 region of the trp promoter. The p L promoter from bacteriophage λ. The gene 10 promoter from bacteriophage T7. Each of these promoters interacts with regulatory proteins which provide a controlable switch for either turning on or turning off specific transcription of adjacent cloned gene.

7. 7 In absence of lactose (carbon source): promoter is repressed (turned off) by lac repressor protein In presence of lactose or IPTG (Inducers): promoter active (turn on) In presence of CAP (catabolite activator protein) to the promoter region: increase affinity if the promoter for RNA polymerase, this increase transcription In presence of cAMP (cyclic AMP): Affinity of CAP towards the promoter is increased, thus highest level of transcription In lowest amount of glucose: cAMP level highest

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9. The p L promoter is controlled by the cI repressor protein from bacteriophage λ. A temperature-sensitive mutant of the cI repressor, cI857, is generally used to regulate p L -directed transcription. Cells are first grown at 28 - 30ºC, at which the cI repressor prevent transcription. When the cell culture reaches the desired stage of growth, the temperature is shifted to 42ºC. The cI repressor is inactivated and transcription can proceed.

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11. The gene 10 promoter from bacteriophage T7 requires T7 RNA polymerase for transcription. T7 RNA polymerase gene is inserted in the E. coli chromosome on a bacteriophage T7 lysogen under the control of the E. coli lac promoter. In the absence of IPTG, the lac repressor represses the synthesis of T7 RNA polymerase. Therefore, the target gene is not transcribed. When lactose or IPTG is added to the medium, it binds to the lac repressor. T7 RNA polymerase is transcribed and the target gene is transcribed.

12. When the portion of the spacer region from the E. coli lac promoter was mutated, the activity increased >40 fold in the absence of CRP. The -20 to -13 region was altered from GC-rich to an AT-rich.

13. Increasing Protein Production Plasmid pCP3 was created in an effort to obtain the highest possible level of foreign-protein production in a recombinant E. coli strain. It contains the strong p L promoter, ampicillin resistance gene, a multiple cloning sequence immediately downstream from the promoter, and a temperature- sensitive origin of replication. The plasmid’s copy number increases 5- to 10- fold when the temperature is increased to 42ºC.

14. At lower temperature, the cI repressor, integrated to E. coli chromosomal DNA, is functional. The p L promoter is turned off, and the plasmid copy number is normal. At higher temperature, the cI repressor is inactivated, the p L promoter is active, and the plasmid copy number increases to around 600 copies per cell, increasing protein production.

15. Metabolic Load The over-expression of a foreign protein prevents cell from obtaining sufficient energy and resources for its growth and metabolism so that it is less able to grow rapidly and attain high density.

16. Metabolic Load An increasing plasmid copy number and/or size requires increasing amounts of cellular energy for plasmid replication and maintenance.

17. Minimize the Metabolic Load The metabolic load can be decreased by using a low- copy number rather than a high-copy-number plasmid vector or integration the foreign DNA directly into the chromosomal DNA of the host organism. The use of strong but regulatable promoters is also an effective means of reducing the metabolic load. Completely or partially synthesizing the target gene to better reflect the codon usage of the host organism. Accept a modest level of foreign-gene-expression- perhaps 5% of the total cell protein-and instead focus on attaining a high host cell density.

18. 18 Insert stability A plasmid imposes a metabolic load. High-copy number plasmid impose greater metabolic load than low-copy number plasmid. Cells without plasmid grow faster than cells containing plasmid. Plasmids can be transferred to other microorganisms. SOLUTION: Integrate the plasmid in the genome via recombination! Use episome! For recombination to happen usually a 50 nt region in the insert is kept homologous to the destination.

19. 19 Double point cross over Integrates only the portion of interest. The region that is kept within the homologous sequence is exchanged!

20. 20 In case of single crossover that incorporates the entire input plasmid into the host chromosome. Single point cross over

21. 21 Removing Selectable Marker Gene Marker genes are used for selection only. Once selection is done they are no longer important Problems with marker genes: Marker genes are mostly Antibiotic resistant gene. So if they are transferred to other disease causing bacteria by HGT (Horizontal Gene Transfer) they may cause antibiotic resistance in them !

22. 22 How to remove selectable marker: Two step process Select the bacteria first and then introduce your gene of interest in the bacteria in the next step so that by recombination Target gene replaces the marker!!

23. 23 How to remove selectable marker: Cre-loxP recombination system

24. 24 Marker gene is flanked by certain 34dp long, asymetric DNA sequence called loxP. The marker gene along with its flanking region gets integrated into the host chromosomal DNA. Transformed are selected with the aid of antibiotic selection. The marker gene then may be excised by treatment of the Cre enzyme that recognize the flanking region which it excise. Remember Cre synthesis can be inducible!!