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| Line 7: |
Line 7: |
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| | ===Design Notes=== | | ===Design Notes=== |
| − | Cloning strategy:
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| − | N- CsgA-CBD –C (CsgA protein at the N-terminus, which is covalently linked to a cellulose binding domain [CBD] at the C-terminus)
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| − | CsgA source:
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| − | Part: BBa_K1361002 (https://parts.igem.org/Part:BBa_K1361002)
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| − | Curli Fiber generator where CsgBtrunc, a dissociative nucleator, under the control of Pbad promoter
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| − | (will only use as a template for amplifying up CsgA)
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| − | Location: not deposited?
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| − | Total length to be amplified: 453 bp
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| − | DNA sequence: (highlighted region is csgA without the stop codon)
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| − | >BBa_K1361002 Part-only sequence (1609 bp)
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| − | acattgattatttgcacggcgtcacactttgctatgccatagcaagatagtccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccataccgtttttttgggctagctactagagaaagaggagaaatactagatgaaaaacaaattgttatttatgatgttaacaatactgggtgcgcctgggattgcagccgcagcaggttatgatttagctaattcagaatataacttcgcggtaaatgaattgagtaagtcttcatttaatcaggcagccataattggtcaagctgggactaataatagtgctcagttacggcagggaggctcaaaacttttggcggttgttgcgcaagaaggtagtagcaaccgggcaaagattgaccagacaggagattataaccttgcatatattgatcaggcgggcagtgccaacgatgccagtatttcgcaaggtgcttatggtaatactgcgatgattatccagaaaggttctggtaataaagcaaatattacacagtatggtactcaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagagttgacagctagctcagtcctaggtattgtgctagctactagagtcacacaggaaagtactagatgaaacttttaaaagtagcagcaattgcagcaatcgtattctccggtagcgctctggcaggtgttgttcctcagtacggcggcggcggtaaccacggtggtggcggtaataatagcggcccaaattctgagctgaacatttaccagtacggtggcggtaactctgcacttgctctgcaaactgatgcccgtaactctgacttgactattacccagcatggcggcggtaatggtgcagatgttggtcagggctcagatgacagctcaatcgatctgacccaacgtggcttcggtaacagcgctactcttgatcagtggaacggcaaaaattctgaaatgacggttaaacagttcggtggtggcaacggtgccgcagttgaccagactgcatctaactcctccgtcaacgtgactcaggttggctttggtaacaacgcgaccgctcatcagtactaatacatcatttgtattacagaaacagggcgcaagccctgttttttttcgggagaagaatatgaatacgttattactccttgcggcactttccagtcagataacctttaatacgacccagcaaggggatgtgtataccattattcctgaagtcactcttactcaatcttgtctgtgcagagtacaaatattgtccctgcgcgaaggcagttcagggcaaagtcagacgaagcaagaaaagaccctttcattgcctgctaatcaacccattgctttgacgaagttgagtttaaatatttccccggacgatcgggtgaaaatagttgttactgtttctgatggacagtcacttcatttatcacaacaatggccgccctcttcagaaaagtcttaa
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| − |
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| − | However, we will use E. coli strain Nissle genomic DNA as a template to PCR and clone out the csgA gene (without the stop codon).
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| − | Nissle csgA DNA sequence:
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| − | >E_coli_Nissle1917_csgA_GNBM_3958
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| − | atgAAACTTT TAAAAGTAGC AGCAATTGCA GCAATCGTAT TCTCCGGTAG CGCTCTGGCAGGTGTTGTTC CTCAGTACGG CGGCGGCGGT GGCAACCACG GTGGTGGCGG TAATAACAGCGGTCCGAATT CAGAGCTGAA TATTTACCAG TACGGTGGCG GTAACTCTGC TCTTGCTCTGCAAGCTGACG CCCGTAACTC TGATCTGACC ATTACCCAGC ACGGCGGCGG TAATGGCGCAGATGTGGGCC AAGGTTCTGA TGACAGCTCA ATCGATCTGA CTCAGCGTGG TTTCGGCAACAGCGCTACTC TTGATCAGTG GAATGGTAAA GATTCTACTA TGACTGTTAA ACAGTTCGGTGGCGGTAACG GTGCTGCTGT TGACCAGACT GCATCTAACT CCAGCGTTAA CGTCACTCAGGTTGGCTTTG GTAACAACGC GACCGCTCAT CAGTACtaa (“taa” is the stop codon)
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| − | AA sequence:
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| − | (frame 1)
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| − | M K L L K V A A I A A I V F S G S A L A G V V P Q Y G G G G G N H G G G G N N S G P N S E L N I Y Q Y G G G N S A L A L Q A D A R N S D L T I T Q H G G G N G A D V G Q G S D D S S I D L T Q R G F G N S A T L D Q W N G K D S T Met T V K Q F G G G N G A A V D Q T A S N S S V N V T Q V G F G N N A T A H Q Y
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| − |
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| − | CBD source:
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| − | Part: BBa_K1321340 (https://parts.igem.org/Part:BBa_K1321340:Design)
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| − | Double CBD (dCBD) with N-terminal linker.
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| − | Location: 2017 Kit Plate 5, well 18A (https://parts.igem.org/partsdb/get_part.cgi?part=BBa_K1321340)
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| − | Total length: 366 bp
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| − | DNA sequence:
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| − | >BBa_K1321340 Part-only sequence (366 bp)
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| − | cccggcgccaatccgcccggtactacgaccacttcgcgtccagcaaccacgaccgggtcgagccccggcccacaggcctgttcttccgtctggggtcagtgcggcggacagaactggtctggccctacctgctgtgcttctggcagcacatgcgtctactctaacgactactatagccagtgcctgccgggtgctaacccgccaggcactaccactacctctcgccccgctaccactacaggctctagcccgggacctacccaaagccactacggccagtgcggaggcattggctacagcggccctaccgtctgcgcctccggaaccacctgccaagtcctgaacccgtactactcccagtgtctt
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| − |
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| − | AA sequence:
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| − | (frame 1)
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| − | P G A N P P G T T T T S R P A T T T G S S P G P Q A C S S V W G Q C G G Q N W S G P T C C A S G S T C V Y S N D Y Y S Q C L P G A N P P G T T T T S R P A T T T G S S P G P T Q S H Y G Q C G G I G Y S G P T V C A S G T T C Q V L N P Y Y S Q C L
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| − |
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| − | Additional info:
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| − | (CBD)
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| − | http://2014.igem.org/Team:Imperial/Functionalisation
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| − | https://parts.igem.org/Part:BBa_K1321340
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| − | https://parts.igem.org/cgi/assembly/plates.cgi?id=4993
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| − | (from https://parts.igem.org/Part:BBa_K1321340)
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| − | This part is based on the double cellulose-binding domain construct (CBDcbh2-linker-CBDcbh1) synthesised and characterised by Linder et al (1) who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.
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| − | The two CBDs are from the fungus T. reesei (Hypocrea jecorina) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder et al (1). The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the CBM family 1. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder et al (1); we chose to use the sequence from the paper since the protein was expressed and characterised successfully.
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| − | References
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| − | 1. Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full
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| | ===Source=== | | ===Source=== |
