Difference between revisions of "Part:BBa K3971028:Design"

 
 
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
blah
+
In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 (upstream) and BamH1 (downstream) flank it, so that the promoter can be easily excised out and replaced with another and the effect of different promoters on sucrose production efficiency can be studied. Two different restriction sites were chosen in order to allow for directional cloning of the promoter. Thus any promoter that needs to be inserted into this cassette would need to be flanked by Nde1 and BamH1 in order to ligate in a directional manner.
 +
 
 +
Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes. Since this is a linear cassette, in order to cut it using Kpn1, whose recognition sites are at the ends of the cassette, 3 bp sequences were added to allow for efficient cleavage by Kpn1 at the ends.
  
  
Line 13: Line 15:
 
===Source===
 
===Source===
  
−
blah
+
The terminator (BBa_B0015) is available in the iGEM distribution kit. PcpcB-m6 was generously provided to us by the Wangikar Lab at IIT Bombay. The restriction enzyme sites are incorporated into the cassette by adding them in primers. The homology arms for Neutral Site 3 and the sps gene can be amplified from the <i>S. elongatus</i> UTEX 2973 genome.
  
 
===References===
 
===References===

Latest revision as of 19:43, 20 October 2021


Cassette to insert sps gene in the Neutral Site 3 of the S. elongatus UTEX 2973 genome.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2877
    Illegal PstI site found at 644
    Illegal PstI site found at 2915
    Illegal PstI site found at 3636
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2877
    Illegal NheI site found at 3070
    Illegal PstI site found at 644
    Illegal PstI site found at 2915
    Illegal PstI site found at 3636
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2877
    Illegal BamHI site found at 1196
    Illegal XhoI site found at 2654
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2877
    Illegal PstI site found at 644
    Illegal PstI site found at 2915
    Illegal PstI site found at 3636
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2877
    Illegal PstI site found at 644
    Illegal PstI site found at 2915
    Illegal PstI site found at 3636
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 (upstream) and BamH1 (downstream) flank it, so that the promoter can be easily excised out and replaced with another and the effect of different promoters on sucrose production efficiency can be studied. Two different restriction sites were chosen in order to allow for directional cloning of the promoter. Thus any promoter that needs to be inserted into this cassette would need to be flanked by Nde1 and BamH1 in order to ligate in a directional manner.

Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes. Since this is a linear cassette, in order to cut it using Kpn1, whose recognition sites are at the ends of the cassette, 3 bp sequences were added to allow for efficient cleavage by Kpn1 at the ends.


Source

The terminator (BBa_B0015) is available in the iGEM distribution kit. PcpcB-m6 was generously provided to us by the Wangikar Lab at IIT Bombay. The restriction enzyme sites are incorporated into the cassette by adding them in primers. The homology arms for Neutral Site 3 and the sps gene can be amplified from the S. elongatus UTEX 2973 genome.

References