gaaccactca gggtcctgtg gacagctcac gaattcctag ctgcaatggc tacaggctcc cggacgtccc tgc

Question

gaaccactca gggtcctgtg gacagctcac gaattcctag ctgcaatggc tacaggctcc

cggacgtccc tgctcctggc ttttggcctg ctctgcctgc cctggcttca agagggcagt

gccttcccaa ccattccctt atccaggctt tttgacaacg ctatgctccg cgcccatcgt

ctgcaccagc tggcctttga cacctaccag gagtttgaag aagcctatat cccaaaggaa

cagaagtatt cattcctgca gaacccccag acctccctct gtttctcaga gtctattccg

acaccctcca acagggagga aacacaacag aaatccaacc tagagctgct ccgcatctcc

ctgctgctca tccagtcgtg gctggagccc gtgcagttcc tcaggagtgt cttcgccaac

agcctggtgt acggcgcctc tgacagcaac gtctatgacc tcctaaagga cctagaggaa

ggcatccaaa cgctgatggg gaggctggaa gatggcagcc cccggactgg gcagatcttc

aagcagacct acagcaagtt cgacacaaac tcacacaacg atgacgcact actcaagaac

tacgggctgc tctactgctt caggaaggac atggacaagg tcgagacatt cctgcgcatc

gtgcagtgcc gctctgtgga gggcagctgt ggcttctagc tgcccgggtg gcatccgaat

tcctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc

agccttgtcc taataaaatt aagttgcatc aaaa

I would like to insert a fragment of this DNA into a plasmid, so that I can express the encoded protein in bacteria. I have a plasmid that can be cut with a restriction enzyme, which can then be used to insert the gene. The only two restriction enzymes that can be used to cut the plasmid are either PstI or EcoRI.

            Use the online webcutter 2.0 site to identify restriction sites in the DNA sequence above, and highlight, in the sequence above, the EcoRI and PstI sites. Use different highlights to distinguish different enzyme cuts sites and the start and stop codons.

1pt

e. Which of the two enzymes should I use to cut the gene sequence and the plasmid in order to insert the entire open reading frame into the plasmid? Briefly explain your answer.

1pt

e. If I can express the human protein in bacterial cells, and purify it, indicate two uses for it.

Explanation / Answer

In order to solve this problem we find out the ORF and check from where the ORF is starting and where it end.

DNA sequence

GAACCACTCAGGGTCCTGTGGACAGCTCACGAATTCCTAGCTGCAATGGCTACAGGCTCCCGGACGTCCCTGCTCCTGGCTTTTGGCCTGCTCTGCCTGCCCTGGCTTCAAGAGGGCAGTGCCTTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAGCTGCCCGGGTGGCATCCGAATTCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCAAAA

In this sequence ATG is representing the ORF GAATTC represent the Eco RI site, CTGCAG represent the Pst I restriction site and TAG represent the termination codon. Here Eco RI site completing the complete ORF hence this can be use for cloning.

This protein having following sequence

MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

When Blast P done then this protein is showing maximum maching with somatotropin hormone. This protein resembles with human somatotropin growth hormone.

This protein is used in those patients who unable to gain natural growth especially childrens.

This protein also use to treat Noonan, turner syndrome and kidney failure.

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