(A) In-Fusion Cloning 75 points total Human caveolin-2 (Cav2) and caveolin-3 (Ca

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Question

(A) In-Fusion Cloning 75 points total Human caveolin-2 (Cav2) and caveolin-3 (Cav3) are homologous membrane proteins which are involved in development of certain types of cancers if their cellular expression is misregulated. A Caveolin consists of two domains-the N- terminal and the C-terminal-each serving a different functional role. To develop a viable target for pharmaceutical intervention the interactions of Cav2 and Cav3 with neighboring proteins and each other need to be understood. The research project requires construction and expression of a 150 amino acid chimera protein that has the N-terminal domain of Cav2 and the C-terminal domain of Cav3. For this, you will need to clone 42 C-terminal residues of Cav3 after the 108 N terminal residues of Cav2. Two plasmids are available, one is an expression vector encoding Cav2, and another is a cloning vector encoding Cav3. Cav-2 DNA sequence atggggetqg agacqgagaa ggeggacgta cacctctt… twacgacca c-cctacage caccacagcq quetcqagta cccccaccce qaaaaattcg aggacqtcat cacaqageca qtgactacac actcctttna caaaqtqtgg atctacaacc atnecctem tgaaatcage aaatacataa tat.aca antt cc-cacagtq ttcctqq22a ttcccc.cc ct-cattgcq gga attctct tt coa ccct cacctatc to cacatctgga ttttaat-cc ttttgtaaaa acctacetaa gtte-acc ttcagtoeag acaatatgga aasas san agRAGE EAte att gatGGar gtgGAGgag GggaggagggR..SE.. e555cacce: geNactgage eaggattaa Cav-3 DNA sequence atc atcccagaag aqcacacaga tctcqaggcc cacatCqtca aquatateca ctacaaggag attgacctgg tqaaccara ccccaaoaac attaacaagg acataqtcaa aqtgga tttt gaagacqtqa tcgeagag^^ tqtqngeace tacagettta acnnngtqtg gaagqtgage tac accacct teact^-^ caagtactgg tastaccatc ageeawGAR ARSEggga ggARGARogg GGG58SEe54 gggg..ggrg,AAGSEgea tc-cc-tecg ccacatctgg gcagtagtac catscattaa cacctaccto ategaga tee ag_ncatca^ ccacatetac tcastctga tccgcacct-exacaaccca ctettcgcqg ccctngccca aqtetasage ageatcaagg tagtnetacq naaggaggte taa

Explanation / Answer

Answer 1. For In-fusion cloning technique we will use pCDNA3.1 (+) vector

For cloning purpose, we will KpnI site will be used in Cav2 sequence and XhoI site will be used in Cav3 primer.

Answer 2. Restriction enzyme site is present in the multiple cloning sites (MCS

Answer 3.

Oligo 1 (KpnI) (Insert 1) Forward – AGCGTTTAAACTTAAGCTTGGTACCATGGGGCTGGAGACGGAG

Oligo 2 (Insert 1) Reverse- CTGCCATCATGCTGAGGGTGGCAAAGAG

Oligo 3 (Insert 2) Forward - CACCCTCAGCATGATGGCAGAAGAGCACAC

Oligo 4 (Insert 2) (XhoI) Reverse – ACGGGCCCTCTAGACTCGAGTTCAAAATCCACCTTGACTATGTCC sequence underlined is vector specific and nucleotide without underlined are insert specific

Answer 4.

Length (bp)

GC%

Tm (Â°C)

Gene Specific Tm (Â°C)

Melting temperature difference

74.2

63.4

Between the forward and reverse primer is 3.2

71.1

60.5

73.2

Between the forward and reverse primer is 4.4

68.8

61.2

Answer 5. Final nucleotide sequence is

ATGGGGCTGGAGACGGAGAAGGCGGACGTACAGCTCTTCATGGACGACGACTCCTACAGCCACCACAGCGGCCTCGAGTACGCCGACCCCGAGAAGTTCGCGGACTCGGACCAGGACCGGGATCCCCACCGGCTCAACTCGCATCTCAAGCTGGGCTTCGAGGATGTGATCGCAGAGCCGGTGACTACGCACTCCTTTGACAAAGTGTGGATCTGCAGCCATGCCCTCTTTGAAATCAGCAAATACGTAATGTACAAGTTCCTGACGGTGTTCCTGGCCATTCCCCTGGCCTTCATTGCGGGAATTCTCTTTGCCACCCTCAGCATGATGGCAGAAGAGCACACAGATCTCGAGGCCCAGATCGTCAAGGATATCCACTGCAAGGAGATTGACCTGGTGAACCGAGACCCCAAGAACATTAACGAGGACATAGTCAAGGTGGATTTTGAA

ATG is the translation start site

Answer 6.

Translated amino acid

M G L E T E K A D V Q L F M D D D S Y S H H S G L E Y A D P E K F A D S D Q D R D P H R L N S H L K L G F E D V I A E P V T T H S F D K V W I C S H A L F E I S K Y V M Y K F L T V F L A I P L A F I A G I L F A T L S M M A E E H T D L E A Q I V K D I H C K E I D L V N R D P K N I N E D I V K V D F E

Amino acids of Cav2

Met G L E T E K A D V Q L F Met D D D S Y S H H S G L E Y A D P E K F A D S D Q D R D P H R L N S H L K L G F E D V I A E P V T T H S F D K V W I C S H A L F E I S K Y V MetY K F L T V F L A I P L A F I A G I L F A T L S Met

Amino acid of Cav3 Met A E E H T D L E A Q I V K D I H C K E I D L V N R D P K N I N E D I V K V D F E

please write the sequence of FliS in the comment section. It is impossible to copy the sequence from JPEG image.