Team:UCLA/Notebook/Protein Cages/24 June 2015
Phillip's notes
Introduction: Today will be focused on designing the complete constructs to be ordered. The sequence from Yen will be used. They will be designed with the aid of Fasih, in order to utilize Benchling efficiently. The best candidates to be ordered will also be selected, and if time permits, design of primers for the remaining ones will begin.
-Attempt without Fasih The sequence from Yen was translated using ExPASy to
MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGARVITYDRR GFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAV AFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRIS EEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARV FHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIP SGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQR RRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMG AVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH-
Where – signifies stop.
This was then backtranslated in Gene Designer to avoid EcoRI, NotI, XbaI, SpeI, and PstI.
These were the results. With an added TGA for stop. Note the C-terminal HisTag!
>PCquad DNA sequence
ATGCCGTTTATTACCGTGGGCCAAGAAAATTCTACGTCTATTGATTTGTATTATGAGGACCATGGTACCGGTACGCCGGTGGTTCTCATCCACGGCTTTCCGCTATCAGGACATTCTTGGGAGCGTCAGAGCGCTGCGCTTTTAGATGCCGGTGCTCGTGTAATAACGTACGATAGACGCGGTTTTGGCCAGAGCTCTCAGCCAACGACTGGATACGATTATGACACCTTCGCCGCCGATTTAAATACTGTTCTGGAAACCCTGGATCTTCAGGATGCGGTCTTAGTTGGTTTTAGTATGGGCACAGGTGAAGTTGCCCGCTACGTCAGTTCTTATGGCACTGCTCGTATTGCAGCGGTAGCTTTTCTGGCTAGCTTAGAACCCTTTCTATTAAAAACCGATGATAATCCGGATGGGGCGGCTCCACAGGAGTTTTTCGACGGAATTGTGGCCGCTGTGAAAGCCGATAGATATGCTTTCTATACTGGGTTTTTCAATGACTTCTATAATTTAGATGAAAACCTGGGAACACGCATCTCGGAAGAGGCTGTACGGAACTCATGGAATACTGCGGCATCTGGCGGATTCTTTGCTGCCGCAGCCGCGCCGACCACTTGGTATACAGATTTTCGTGCGGACATTCCTAGAATTGATGTACCTGCCCTGATTCTGCACGGTACGGGCGACCGTACCTTACCGATTGAGAACACTGCCCGCGTCTTTCATAAAGCTCTTCCGTCCGCTGAGTACGTAGAGGTTGAAGGGGCACCTCATGGTCTGTTATGGACTCATGCTGAAGAAGTCAACACAGCCCTGCTTGCTTTCCTTGCGAAGGCTCAAGAAGCCCAAAAGCAGAAATTACTGACCGAAGTGGAAACTTATGTACTATCCATCATACCGTCTGGTCCCCTTAAAGCAGAAATCGCACAACGTCTGGAGGATGTCTTCGCGGGCAAAAATACAGACCTGGAGGTGCTGATGGAATGGCTTAAGACCCGACCGATTCTGTCACCGTTAACGAAGGGTATCCTAGGATTCGTTTTTACCCTGACGGTGCCCAGTGAGCGCGGACTGCAACGAAGAAGATTCGTCCAAAACGCATTAAACGGGAATGGTGACCCAAACAATATGGACAAAGCGGTGAAGCTGTATCGAAAACTTAAGCGCGAAATAACATTCCATGGGGCCAAAGAAATTAGCCTGAGCTATTCCGCTGGGGCGCTTGCTTCTTGTATGGGTTTAATATATAACCGAATGGGTGCGGTCACCACGGAAGTCGCGTTCGGATTAGTATGCGCGACATGCGAGCAGATTGCAGACTCTCAGCATAGAAGCCATCGCCAGTTAGAACATCATCACCATCATCATTGA
>PCquad amino acid sequence MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGARVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH The sequence was then annotated on Benchling. Using PDB ID: 1bro and 1aa7 (bromoperoxidase and M1 protein), the linker was identified.
Next, the full insertion sequence was also generated on Gene Designer. >Mutant Cage Full Insertion GGCCTGGTTCCCCGCGGTTCAGGC >Full Insertion GLVPRGSG This allowed easy insertion of the sequences. The full sequence was inserted where needed after removing redundant bases.
For the prefix and suffix, the sequence was obtained from this site. The first prefix sequence was used. http://parts.igem.org/Help:BioBrick_Prefix_and_Suffix prefix-NdeI-gene-XhoI-Histag-suffix
Concluding remarks: Before the rest of the sites are revisited, a meeting will be set up with David and/or Fasih to check our design rationale.