Difference between revisions of "Part:BBa K1465206:Design"

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===Design Notes===
 
===Design Notes===
  
−
*Amplification of the pSB1C3-Backbone using specific the 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]:<br>
+
*Amplification of the pSB1C3-Backbone (<bbpart>BBa_J00450</bbpart>) 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]:<br>
  
 
  '''pSB1C3_pre_csoS4A (47 bp)'''<br>
 
  '''pSB1C3_pre_csoS4A (47 bp)'''<br>
−
5'-ATTCGCGGCCGCTTCTAGAGAAATCATGCAAGTTGAGAAAACG-3'<br>
 
−
'''pSB1C3_suf_csoS4A (47 bp)'''<br>
 
 
  5'-AACGTTTTCTCAACTTGCATGATTTCTCTAGAAGCGGCCGCGAATTC-3'<br>
 
  5'-AACGTTTTCTCAACTTGCATGATTTCTCTAGAAGCGGCCGCGAATTC-3'<br>
 +
'''pSB1C3_suf_csoS4A (46 bp)'''<br>
 +
5'-GTGTATCGCAAATCTGAATAGCTAATACTAGTAGCGGCCGCTGCAG-3'<br>
  
−
*Amplification of the csoS1D containing also the natural interspace with the RBS using specific the Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS1D_fwd csoS1D_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS1D_rev csoS1D_rev]:<br>
+
*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:<br>
  
−
  '''csoS1D_fwd (40 bp)'''<br>
+
  '''csoS4A_fwd (47 bp)'''<br>
−
  5'-ATTCGCGGCCGCTTCTAGAGTGTCTAGTAGGGAAGATGCG-3'<br>
+
  5'-ATTCGCGGCCGCTTCTAGAGAAATCATGCAAGTTGAGAAAACG-3'<br>
−
  '''csoS1D_rev (41 bp)'''<br>
+
  '''csoS4A_PstI-rev (43 bp)'''<br>
−
  5'-TGCAGCGGCCGCTACTAGTATTAGAACCCTTCAGCGCGACG-3'<br>
+
  5'-GAGGGATAAGACTTGCTTCCGGCAGCCTCTCGTGCTGCCGATG-3'<br>
  
 
*[http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone and the coding sequence of pore-froming protein csoS1D containing also the natural interspace region with RBS from ''Halothiobacillus neapolitanus''.<br>
 
*[http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone and the coding sequence of pore-froming protein csoS1D containing also the natural interspace region with RBS from ''Halothiobacillus neapolitanus''.<br>

Revision as of 18:58, 3 October 2014


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'
  • [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone and the coding sequence of pore-froming protein csoS1D 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.]