Iago's Motives In Othello Essay

2034 words - 8 pages

Ruth Frenkel MIC 220 Challenge questions set #2 Due April 3, 2002 SID# 601-50-5380 1.2. There are a multitude of reasons that the Escherichia coli did not produce the interferon after being transformed. First, the E. coli cells might not have accepted the plasmid during the transformation process. If the electroporation was used, the voltage might have been too high and irreversibly damaged the cell membrane, thereby rendering the cells incompetent. Conversely, the voltage used might have been too low and not have induced micropores in the cell membrane large enough for the uptake of foreign DNA. For E. coli the voltage range for successful transformation by electroporation is 250 to 4,000 V/cm. Voltages above or below this range will not result in transformed cells, and thus will not express interferon.Another explanation for the lack of interferon production is that the plasmid did not contain a strong promoter upstream of the inserted interferon gene. Without a strong promoter, the E. coli's RNA polymerase will not bind to the recombinant plasmid and thus will not transcribe the interferon gene. On the other hand, if there was a strong promoter, the protein could have been over-transcribed and the host cells machinery would be overloaded, resulting in inadequate expression of the interferon.Yet another reason for the absence of interferon expression is that the inserted gene fragment did not have a leader sequence, also known as a signal peptide. These sequences are typically located at the N-terminus of a protein and facilitate the transport of expressed proteins into the periplasm. This exportation renders the expressed foreign protein more stable than if it remains in the E. coli's cytoplasm. If the interferon gene was not attached to a signal peptide, then it may have been degraded in the cytoplasm by endogenous exonucleases.Also, since interferon is a eukaryotic protein, its pre-mRNA sequence will contain introns that need to be excised before proper translation into the protein occurs. Since E. coli is a prokaryote, it does not possess the necessary machinery, the spliceosome, to splice out the introns and then translate the mRNA sequence into the protein. If the introns are not excised, the resultant protein will not be interferon but rather a nonsense peptide of unknown function. One way to circumvent this aberration is to synthesize cDNA from already processed mRNA in the laboratory before inserting the sequence into the plasmid. This ensures that only the coding regions of the gene are being transcribed and will be translated into the correct amino acid sequence. Another reason could be that the growth temperature for the bacterial culture was not optimal, thus inhibiting growth of the E. coli cells and not allowing for the expression of exogenous proteins. E. coli's optimal temperature range is between 28 C and 40 C, with optimal growth achieved at 37 C. Any temperature above this range will denature essential...

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