V. HOMEWORK

Your goal is to recombinantly produce of 2 g of highly pure haloalkane dehalogenase DhaA enzyme
from Desulfobacterium autotrophicum. See amino acid and corresponding nucleotide sequences below:

>ACN15444.1 DhaA [Desulfobacterium autotrophicum HRM2]

MVTRDPAEQSRNIKSPGIRRKINGTMVGTKDFYEIYPFVPHFMTLDRHKLHYLDLGKGSPVVMVHGNPTWSFYFRRLARDLSVNHRVIVPDHMGCGLSD
KPSTRDYDYTLASRVRDLDRLIQSLDLGKKITLVVHDWGGMIGCAWALRHLDRIDRIIITNTSGFHLPGAKRFPLRLWLIKYLPWFAIPGIQGLNLFAR
AALYMAPKQSLSTTVRQGLTAPYNSWKNRIATLKFVQDIPLSPRDKSYELVNWVDTHLEGLKTVPMMILWGRHDFVFDLSFLDEWNKRFPHAQTHIFED
AGHYLFEDKPDETSNLIKKFIEEY

>CP001087.1:2679075-2680040 Desulfobacterium autotrophicum HRM2, complete genome

ATGGTAACCAGGGATCCAGCGGAGCAAAGCAGAAACATCAAAAGTCCGGGCATCAGAAGAAAGATCAACGGCACCATGGTCGGCACCAAGGATTTTTAT
GAAATATATCCCTTTGTTCCCCATTTCATGACCCTGGACCGGCACAAACTCCACTACCTTGACCTGGGTAAGGGAAGTCCAGTTGTCATGGTCCACGGT
AATCCCACCTGGTCGTTTTATTTTCGCAGGCTTGCCCGGGATCTTTCGGTGAACCACCGGGTCATTGTTCCCGACCACATGGGGTGCGGCCTGTCTGAC
AAGCCGTCCACCAGGGATTACGACTATACCCTTGCATCAAGGGTCCGGGACCTGGACCGTCTGATCCAGAGCCTTGACCTTGGAAAAAAGATCACCCTG
GTCGTCCACGACTGGGGCGGTATGATCGGCTGCGCCTGGGCCCTTCGTCACCTGGACAGGATAGACAGGATCATCATCACCAACACCTCGGGGTTTCAT
CTTCCCGGGGCAAAACGATTTCCCCTGCGGCTTTGGCTGATCAAATACCTTCCCTGGTTTGCCATTCCAGGGATTCAGGGCCTGAATCTCTTTGCCAGG
GCAGCCCTTTACATGGCTCCGAAACAATCACTTTCAACAACGGTCAGGCAGGGGCTCACGGCACCCTACAACTCGTGGAAAAACAGGATCGCCACCCTC
AAATTTGTCCAGGACATTCCCCTTTCACCCAGGGACAAAAGCTACGAACTTGTCAACTGGGTGGACACCCACCTTGAAGGTCTTAAAACCGTTCCCATG
ATGATCCTATGGGGCAGACACGATTTTGTGTTTGATCTGTCGTTCCTTGACGAGTGGAACAAACGGTTTCCCCATGCCCAAACACATATTTTCGAGGAT
GCAGGCCATTATCTGTTTGAGGACAAACCCGATGAAACATCAAATCTTATCAAAAAATTCATAGAGGAGTACTAA

1.       Select a suitable expression host (heterologous system) for the DhaA enzyme overproduction
and explain why the selected host is the best choice:








2.       Propose and design a strategy for the DNA template synthesis, including primer design:










3.       Propose a cloning strategy – ligation-dependent versus ligation-independent cloning,
selection of expression vector, affinity/solubility tags etc.? How will you check the error-free
clones?















4.       Briefly describe production process – how will you introduce a foreign gene into the host,
from pre-culture to large-scale overproduction, inducible versus stable expression, cytotoxicity
issue, timing, harvesting strategy etc.














5.       How will you determine the quality and yield of the purified enzyme?
















6.       How will you determine oligomeric state of the DhaA enzyme?