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Title: How do I construct a plasmid Adding GFP using pUC19? Post by: karmahone on Mar 31, 2017 Homework: construct a plasmid to direct expression of this fusion protein (GFP) in E. coli using pUC19. Write down the whole sequence of your plasmid MTWSRRQFLTGVGVLAAVSGTAGRVVA - green fluorescent protein Title: Re: How do I construct a plasmid Adding GFP using pUC19? Post by: habiba on Mar 31, 2017 Following websites would be beneficial to you concerning this assignment -
https://www.addgene.org/mol-bio-reference/plasmid-backround/ www.ncbi.nim.nih.gov >NCBI>Literature>Pub Med Central(PMC) https://en.wikipedia.org/wiki/Vector_(molecular_biology) www.molbiol-tools.ca/molecular-biology-freeware.htm Title: Re: How do I construct a plasmid Adding GFP using pUC19? Post by: 5seal on Mar 31, 2017 Using the standard -35 and -10 sequences for sigma-70 recognition and an ideal consensus Shine-Delgarno site.
Sequence for Promoter: TTGATATNNNNNNNNNNNNNNNNNNTATA ATNNNNNNATG DNA Transcription Sequence (Shine-Delgarno included): ATG(TAAGGAGGT)NNNNNNNNATGACGTGGTCTAGGAGGCAG TTTTTGACGGGGGTGGGGGTGTTGGCGGC GGTGTCTGGGACGGCGGGGAGGGTGGTGG CG (the italized eventually translates to MTWSRRQFLTGVGVLAAVSGTAGRVVA; the polypeptide that will use the TAT system) Spacer DNA sequences between the protein and GFP if needed (four repeats of Ala-Gly-Ser): GCTGGCTCTGCTGGCTCTGCTGGCTCTGC TGGCTCT Finally, GFP: ATGAGTAAAGGAGAAGAACTTTTCACTGG AGTTGTCCCAATTCTTGTTGAATTAGATG GTGATGTTAATGGGCACAAATTTTCTGTC AGTGGAGAG GGTGAAGGTGATGCAACATACGGAAAACT TACCCTTAAATTTATTTGCACTACTGGAA AACTACCTGTTCCATGGCCAACACTTGTC ACTACTTTCTCTTATGGTG TTCAATGCTTTTCAAGATACCCAGATCAT ATGAAACGGCATGACTTTTTCAAGAGTGC CATGCCCGAAGGTTATGTACAGGAAAGAA CTATATTTTTCAAAGATGA CGGGAACTACAAGACACGTGCTGAAGTCA AGTTTGAAGGTGATACCCTTGTTAATAGA ATCGAGTTAAAAGGTATTGATTTTAAAGA AGATGGAAACATTCTTGG ACACAAATTGGAATACAACTATAACTCAC ACAATGTATACATCATGGCAGACAAACAA AAGAATGGAATCAAAGTTAACTTCAAAAT TAGACACAACATTGAAGA TGGAAGCGTTCAACTAGCAGACCATTATC AACAAAATACTCCAATTGGCGATGGCCCT GTCCTTTTACCAGACAACCATTACCTGTC CACACAATCTGCCCTTTC GAAAGATCCCAACGAAAAGAGAGACCACA TGGTCCTTCTTGAGTTTGTAACAGCTGCT GGGATTACACATGGCATGGATGAACTATA CAAATAG The DNA sequences above are simply put together. I also added a couple extra TAA stops on the end to ensure translation termination. A short description: Use same restriction enzyme (perhaps EcoR1) to cut pUC19 and vector insert. Fuse insert strand into vector by mixture of ATP and DNA Ligase. Transform E.coli K12 using calcium chloride heat shock. Plate on ampicillin and X-gal medium to obtain successfully transformed colonies utilizing the manufactured plasmid. |