Part 1: Benchling & In-silico Gel Art

See the Gel Art: Restriction Digests and Gel Electrophoresis protocol for details. Overview:

• [x] Make a free account at benchling.com
• [x] Import the Lambda DNA.
• [x] Simulate Restriction Enzyme Digestion with the following Enzymes: EcoRI-HF HindIII-HF BamHI-HF KpnI-HF EcoRV-HF SacI-HF (if available) SalI-HF (if available)
• [ ] Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks.

Part 3: DNA Design Challenge

<aside> ⚠️ Mandatory for MIT/Harvard Students and Committed Listeners. Due start of recitation Wed Feb 21

</aside>

3.1.     Choose your protein.

In recitation, we discussed that you will pick a protein for your homework that you find interesting. Which protein have you chosen and why? Using one of the tools described in recitation (NCBI, UniProt, google), obtain the protein sequence for the protein you chose.

Deschampsia Antartica is one of the only two members of the Spermatophyta family that inhabits the artic. Because of this the activity of it´s antifreezing protein is a great study subject.

ACN38302.1 ice recrystallization inhibition protein 7 [Deschampsia antarctica]

MAPKCWLLLLFSVLLLPVASATSCHPDDLRALQGFARNLGGVEGVLVRAAWFGASCCSWEGVGCDDATGRVTVLQFPTRGLAGAIPGASLAGLARHVKRNRRTLADQPNTISGTNNHVRSGKNNVISGNDNTVISGDSNTVSGSNNTVTTGSDNTVTGSNHVVSGRNHIVTDNNNAVTGNDNNVSGSFHTVSGSHNTVSRSNNTVSGSNHVVSGSNKVVTGG

3.2.     Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

The Central Dogma discussed in class and recitation describes the process in which DNA sequence becomes transcribed and translated into protein. The Central Dogma gives us the framework to work backwards from a given protein sequence and infer the DNA sequence that the protein is derived from. Using one of the tools discussed in class, NCBI or online tools (google “reverse translation tools”), determine the nucleotide sequence that corresponds to the protein sequence you chose above.

reverse translation of ACN38302.1 ice recrystallization inhibition protein 7 [Deschampsia antarctica] to a 666 base sequence of most likely codons. atggcgccgaaatgctggctgctgctgctgtttagcgtgctgctgctgccggtggcgagc gcgaccagctgccatccggatgatctgcgcgcgctgcagggctttgcgcgcaacctgggc ggcgtggaaggcgtgctggtgcgcgcggcgtggtttggcgcgagctgctgcagctgggaa ggcgtgggctgcgatgatgcgaccggccgcgtgaccgtgctgcagtttccgacccgcggc ctggcgggcgcgattccgggcgcgagcctggcgggcctggcgcgccatgtgaaacgcaac cgccgcaccctggcggatcagccgaacaccattagcggcaccaacaaccatgtgcgcagc ggcaaaaacaacgtgattagcggcaacgataacaccgtgattagcggcgatagcaacacc gtgagcggcagcaacaacaccgtgaccaccggcagcgataacaccgtgaccggcagcaac catgtggtgagcggccgcaaccatattgtgaccgataacaacaacgcggtgaccggcaac gataacaacgtgagcggcagctttcataccgtgagcggcagccataacaccgtgagccgc agcaacaacaccgtgagcggcagcaaccatgtggtgagcggcagcaacaaagtggtgacc ggcggc

3.3.     Codon optimization.

Once nucleotide sequence of your protein is determined, you need to codon optimize your sequence. You may, once again, utilize google for a “codon optimization tool”. In your own words, describe why do you need to optimize codon usage. Which organism have you chose to optimize the codon sequence for and why?

I chose to express the protein in E.coli becaus it doesn´t have complex post-translational modifications so this high expression system can be used. Codon optimization is required to achieve the maximun possible yield in protein production in the desired organism, because of the different tRNA abundances in different organisms. Optimized with genscript free tool:

ATGGCTCCCAAATGTTGGCTTTTATTGCTATTCAGCGTTTTGCTGCTGCCGGTTGCGAGCGCAACCAGCTGCCATCCAGATGACCTGCGCGCGCTGCAGGGTTTTGCACGCAACCTGGGTGGCGTTGAAGGTGTCTTGGTTAGAGCCGCATGGTTTGGTGCGAGCTGCTGCAGCTGGGAGGGTGTTGGTTGTGATGACGCGACCGGCCGTGTTACTGTGTTACAATTCCCGACCCGTGGTTTGGCGGGTGCCATTCCGGGTGCTAGCCTGGCGGGCCTCGCTCGCCACGTGAAACGTAATCGTCGTACCCTGGCGGATCAGCCGAATACCATTAGCGGAACCAATAATCACGTGCGCAGCGGCAAGAACAACGTGATCAGCGGCAATGACAATACCGTCATCAGCGGTGATAGCAACACCGTGTCCGGTTCCAATAACACCGTGACGACCGGCTCAGATAACACTGTTACCGGCTCTAACCATGTTGTCTCGGGCCGTAACCACATCGTGACCGACAATAACAACGCTGTGACAGGTAATGACAACAACGTGTCCGGTTCGTTCCACACCGTATCCGGTTCCCATAATACGGTGAGCCGTTCTAACAACACGGTCAGCGGCTCTAACCACGTTGTGTCTGGCAGTAACAAAGTTGTTACGGGTGGC