Coding

Part:BBa_K2522000:Design

Designed by: Dallas McDonald   Group: iGEM17_US_AFRL_CarrollHS   (2017-09-17)


csgA-dCBD (Curli fibers attachable to cellulose)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

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.

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.

Steps to engineer CsgA-dCBD

  1. PCR amplify with Phusion (a method to make copies of DNA) both csgA and dCBD genes:
    • csgA => use Nissle genomic DNA as template
    • dCBD => use BBa_K1321340 as template (not sure if it comes as DNA or as bacteria carrying the plasmid)
  2. Clone csgA and dCBD into TOPO (pCR-TOPO Blunt).
  3. Transform into TOP10 (or NEB5alpha).
  4. Screen for correct clones and sequence to verify clones.
  5. Clone csgA into pBAD (call the plasmid pBAD-csgA or pAAJM1 [for Andrea/Angela/Jonah/Max]).
  6. Screen for correct clones and sequence to verify clones.
  7. Clone dCBD into pBAD-csgA (or pAAJM1). Name the new plasmid.
  8. Screen for correct clones and sequence to verify clones.
  9. Test for expression of the clone!

Source

Cloning strategy:

N- CsgA-CBD –C (CsgA protein at the N-terminus, which is covalently linked to a cellulose binding domain [CBD] at the C-terminus)

CsgA source:

Part: BBa_K1361002 (https://parts.igem.org/Part:BBa_K1361002)

Curli Fiber generator where CsgBtrunc, a dissociative nucleator, under the control of Pbad promoter (will only use as a template for amplifying up CsgA)

Location:

not deposited?

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).

Nissle csgA DNA sequence:

>E_coli_Nissle1917_csgA_GNBM_3958

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)

AA sequence:

(frame 1)

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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CBD source:

Part: BBa_K1321340 (https://parts.igem.org/Part:BBa_K1321340:Design)

Double CBD (dCBD) with N-terminal linker.

Location:

2017 Kit Plate 5, well 18A (https://parts.igem.org/partsdb/get_part.cgi?part=BBa_K1321340)

Total length:

366 bp


AA sequence:

(frame 1)

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

Additional info:

(CBD)

http://2014.igem.org/Team:Imperial/Functionalisation

https://parts.igem.org/Part:BBa_K1321340

https://parts.igem.org/cgi/assembly/plates.cgi?id=4993

(from https://parts.igem.org/Part:BBa_K1321340)

References

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