You are assigned the task to express an E. coli gene in cultured human cells. Fr

Question

You are assigned the task to express an E. coli gene in cultured human cells. From published research, you know the amino acid sequence of the E. coli protein, which requires no post-translational modification.

2A. How would you modify the coding sequence of the E. coli gene in order to optimize its expression in human cells? This operation is done on a computer, not resulting in actual DNA molecules, but rather a sequence of nucleotides.

The coding sequence of the E. Coli gene must be modified with a Kozak sequence at 5 prime end because it aids in the initiation of translation.

2B. How will you actually produce the optimized DNA sequence in order to clone it in an expression vector for human cells? Describe the methods in conceptual terms. Use as simple a method as possible. Do not skip important steps.

Explanation / Answer

Answer 2A-

Use software algorithm (Nucleotide BLAST) to identify the putative best sequence encoding the same protein in E.coli. That sequence should be analyzed with respect to the following parameters

1-codon equality to avoid codon bias

2-GC content

3-DNA motifs like RBS (Ribosome binding sites)

The ideal sequence must be identical by weighted scoring and penalty system. Using a sliding window from 5'-3' end, optimized full length can be generated by using gene optimizeic algorithms.

Construct designing cDNA from genebank or denovo synthesis of sequence->Optimization of sequence using gene optimization algorithms->Selection of optimized construct expression in cultured human cells- The optimized gene/ construct to be cloned or synthesized in expression vector with T7 promoter, lac operon,RBS, start codon(ATG), 6sHis(N terminal His Tag), amber stop codon,ori site,and canamycin resistance gene->Transform this expression plasmid in to mammalian cell line->check the expression in total cell lysate

Answer 2B-

Synthesize or amplify the required gene/construct. Expression of this gene in mammalian cell requires suitable cell line and supported vector. The most widely used cell lines are Hela cells, myloma cells, Human embryonic kidney. The optimized construct can be cloned into vectors such as Adenovirus or retroviral vectors with efficient promoter and selection markers. Otherwise the construct can be directly transfered in to the cell by electroporation or microinjection.

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