Coding

Part:BBa_K2522000:Design

Designed by: Dallas McDonald   Group: iGEM17_US_AFRL_CarrollHS   (2017-09-17)
Revision as of 19:46, 31 October 2017 by Annie-B (Talk | contribs) (→‎Design Notes)


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 (need a better name than this…)

  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

Genomic sequencing

References