Translational_Unit

Part:BBa_K1465206:Design

Designed by: iGEM-Team Bielefeld 2014   Group: iGEM14_Bielefeld-CeBiTec   (2014-09-29)


Shell-proteins (csoS4AB, csoS1CAB) of the carboxysome of Halothiobacillus neapolitanus


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 704
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

  • Amplification of the pSB1C3-Backbone (BBa_J00450) using the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_csoS4A pSB1C3_pre_csoS4A] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_csoS4A pSB1C3_suf_csoS4A]:
pSB1C3_pre_csoS4A (47 bp)

5'-AACGTTTTCTCAACTTGCATGATTTCTCTAGAAGCGGCCGCGAATTC-3'

pSB1C3_suf_csoS4A (46 bp)

5'-GTGTATCGCAAATCTGAATAGCTAATACTAGTAGCGGCCGCTGCAG-3'
  • Amplification of the front part for the removal of the PstI reconition site. This part contains the natural interspace with the RBS and the front part of the first shell protein csoS4A. For the amplification the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_csoS4A csoS4A_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_csoS4A_PstI csoS4A_PstI-rev] were used:
csoS4A_fwd (47 bp)

5'-ATTCGCGGCCGCTTCTAGAGAAATCATGCAAGTTGAGAAAACG-3'

csoS4A_PstI-rev (43 bp)

5'-GAGGGATAAGACTTGCTTCCGGCAGCCTCTCGTGCTGCCGATG-3'
  • Amplification of the middle part for the removal of the SpeI reconition site. This part contains the rest of the first shell protein csoSA4, the coding sequence of csoS4B, csoS1C, csoS1A and the natural interspaces with the RBS of each. For the amplification the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_PstI_csoS4A PstI_csoS4A_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_SpeI_Intergen SpeI_Intergen_rev] were used:
PstI_csoS4A_fwd (51 bp)

5'-TCATCGGCAGCACGAGAGGCTGCCGGAAGCAAGTCTTATCCCTCTGATTTG-3'

SpeI_Intergen_rev (52 bp)

5'-GTCGTTGCCATAACAGATCCTCTAAAACTAGGTTGAGCACGTTCAGAACGAG-3'
  • Amplification of the last part for the amplicifation of the shell protein csoS1B containing also the natural interspaces with the RBS. For the amplification the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_SpeI_Intergen SpeI_Intergen-fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_csoS1_pSB1C3 csoS1_rev] were used:
SpeI_Intergen-fwd (49 bp)

5'-TCGTTCTGAACGTGCTCAACCTAGTTTTAGAGGATCTGTTATGGCAACG-3'

csoS1_rev (50 bp)

5'-CGGACTGCAGCGGCCGCTACTAGTATTAGCTATTCAGATTTGCGATACAC-3'
  • [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone and the coding sequences of shell proteins csoS4AB and csoS1CAB containing also the natural interspace region with RBS from Halothiobacillus neapolitanus.

Source

  • [http://www.ncbi.nlm.nih.gov/nucleotide/261835099?report=genbank&log$=nuclalign&blast_rank=1&RID=2T27D737014 Halothiobacillus neapolitanus c2 (GenBank: CP001801.1)]: Hneap 0918 (csoS4A)
  • [http://www.ncbi.nlm.nih.gov/nucleotide/261835099?report=genbank&log$=nuclalign&blast_rank=1&RID=2T27D737014 Halothiobacillus neapolitanus c2 (GenBank: CP001801.1)]: Hneap 0917 (csoS4B)
  • [http://www.ncbi.nlm.nih.gov/nucleotide/261835099?report=genbank&log$=nuclalign&blast_rank=1&RID=2T27D737014 Halothiobacillus neapolitanus c2 (GenBank: CP001801.1)]: Hneap 0916 (csoS1C)
  • [http://www.ncbi.nlm.nih.gov/nucleotide/261835099?report=genbank&log$=nuclalign&blast_rank=1&RID=2T27D737014 Halothiobacillus neapolitanus c2 (GenBank: CP001801.1)]: Hneap 0915 (csoS1A)
  • [http://www.ncbi.nlm.nih.gov/nucleotide/261835099?report=genbank&log$=nuclalign&blast_rank=1&RID=2T27D737014 Halothiobacillus neapolitanus c2 (GenBank: CP001801.1)]: Hneap 0914 (csoS1B)
  • 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.]