Translational_Unit

Part:BBa_K1465204:Design

Designed by: iGEM-Team Bielefeld 2014   Group: iGEM14_Bielefeld-CeBiTec   (2014-09-22)


Shell-associated protein (csoS2) of the carboxysome of Halothiobacillus neapolitanus


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 185
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 343
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 851
    Illegal AgeI site found at 1802
    Illegal AgeI site found at 2483
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 243


Design Notes

  • Amplification of the pSB1C3-Backbone (BBa_J04450) using the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_pre_sap_1 pSB1C3_pre_sap] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_sap2 pSB1C3_suf_sap]:
pSB1C3_pre_sap (43 bp)

5'-AGCGCAATGACAGACTTGACCTCTAGAAGCGGCCGCGAATTCC-3'

pSB1C3_suf_sap (40 bp)

5'-CCGGCGGCGCGCGCGGTTAATACTAGTAGCGGCCGCTGCA-3'
  • Amplification of the shell-associated protein csoS2_1 from the Plasmid [http://www.addgene.org/52065/ pHnCBS1D] containing also the natural interspace with the RBS and the front part of the coding sequence using the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS2_sap_1_fwd csoS2_sap_1_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS2_sap_1_rev csoS2_sap_1_rev]:
csoS2_sap_1_fwd (43 bp)

5'-ATTCGCGGCCGCTTCTAGAGGTCAAGTCTGTCATTGCGCTGTG-3'

csoS2_sap_1_rev (38 bp)

5'-GCGCCGAACATGACTTTATCCGCTTGTGAAGGCTTTGC-3'
  • The sequence of the back part from the shell-associated protein csoS2_2 was synthesized using [http://eu.idtdna.com/pages/products/genes/gblocks-gene-fragments gBlocks]. The synthesized framgent does not contain any restriction sites and only the stop codon was optimized for Escherichia coli. The gene fragemnt was amplified using the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#sap_2_fwd csoS2_sap_2_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#sap_2_fwd csoS2_sap_2_rev]:
csoS2_sap_2_fwd (22 bp)

5'-AAGTCATGTTCGGCGCCACAGC-3'

csoS2_sap_2_rev (20 bp)

5'-GCCGCTACTAGTATTAACCG-3'
  • [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone and the coding sequence of shell-associated protein csoS2 (sap) containing also the natural interspace region with RBS from Halothiobacillus neapolitanus.

Source

  • [http://www.ncbi.nlm.nih.gov/nucleotide/261835099?report=genbank&log$=nuclalign&blast_rank=1&RID=2T27D737014 Halothiobacillus neapolitanus c2 (GenBank: CP001801.1)]: Hneap 0920
  • Plasmid [http://www.addgene.org/52065/ pHnCBS1D] from Bonacci et al, 2011

References

  • Walter Bonacci, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver

and David F. Savage (2011) Modularity of a carbon-fixing protein organelle [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258634/pdf/pnas.201108557.pdf Proc Natl Acad Sci U S A. 109: 478–483.]