Plasmid_Backbone
pPIGBACK

Part:BBa_K2350009:Design

Designed by: JO-NING HUNG   Group: iGEM17_NYMU-Taipei   (2017-10-21)
Revision as of 16:48, 22 October 2017 by Yang20051 (Talk | contribs) (→‎Design Notes)


Pigment double-crossover homologous gene recombination bacbone (pPIGBACK)



Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 1
    Illegal suffix found at 3880
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 3881
    Illegal PstI site found at 3895
    Illegal NotI site found at 7
    Illegal NotI site found at 1321
    Illegal NotI site found at 3888
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal BglII site found at 1883
    Illegal BamHI site found at 2180
    Illegal XhoI site found at 1118
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 1
    Illegal suffix found at 3881
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 1
    Plasmid lacks a suffix.
    Illegal XbaI site found at 16
    Illegal SpeI site found at 3881
    Illegal PstI site found at 3895
    Illegal NgoMIV site found at 2101
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 972
    Illegal BsaI site found at 1783


The vector, pPIGBACK, is used to transform into S. elongatus PCC 7942 with the inserted pigment gene in our project. pPIGBACK contains 5’- and 3’-ends of the neutral site II (NSII), replication origin of pBR322, ampicillin resistance gene, and double terminator BBa_B0015. The strategy we chose to construct the vector is to fuse B0015 and AmpR together first. Secondly, we fused 5’- and 3’-ends of the neutral site II (NSII) with PBR322 replication origin (ORI) together. At last, we ligated two parts together. The aim of constructing this vector is to finish double-crossover homologous gene recombination in S. elongatus PCC 7942. To insert BBa_J04450 with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove all Pst1 cutting sites in pPIGBACK.



Design Notes

We use site-directed mutagenesis to remove all Pst1 cutting sites in pPIGBACK. Because pPIGBACK contains four Pst1 cutting sites, we totally mutated four times using site-directed mutagenesis.First site is in ampicillin resistance sequence, second site is in 3NSII sequence, and the last two sites are in 5NSII sequence.


Primers Pst1 site directed mutagenesis of Ampicillin resistance:
GCGTGACACCACGATGCCAGCAGCAATGGCAACAACGTTG
(RC) CAACGTTGTTGCCATTGCTGCTGGCATCGTGGTGTCACGC


Primers Pst1 site directed mutagenesis of 3NSII:
CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA
(RC) TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG


Primers Pst1 site directed mutagenesis of 5NSII:
  1. GGTATGGAAAACGCTTGCTGGAGCATCAGATCGATGCGAT (RC) ATCGCATCGATCTGATGCTCCAGCAAGCGTTTTCCATACC
  2. TTTGGTGGCGGCGATGGCTGGAGAGGCCAGCATTGAAGAC (RC) GTCTTCAATGCTGGCCTCTCCAGCCATCGCCGCCACCAAA

Source

Synechoccocus elongatus PCC7942 genomic DNA and pBR322 nucleotide sequence


References