Difference between revisions of "Part:BBa K1904003"
 
Line 18: Line 18:
  
 
https://static.igem.org/mediawiki/2016/d/da/T--TecCEM--PCR-WELKWIKI.JPG
 
https://static.igem.org/mediawiki/2016/d/da/T--TecCEM--PCR-WELKWIKI.JPG
 +
 +
 
Fig 1.  Colony PCRs of WELK 0.8% agarose gel run at 50 min 100V. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK  Colony 1 PCR  5μL + 4μL LB, 3) WELK  Colony 2 PCR  5μL + 4μL LB, 4) WELK  Colony 3 PCR  5μL + 4μL LB, 5) WELK  Colony 4 PCR  5μL + 4μL LB, 6) WELK  Colony 5 PCR  5μL + 4μL LB, 7) WELK  Colony 6 PCR  5μL + 4μL LB, 8) WELK  Colony 7 PCR  5μL + 4μL LB, 9)WELK  Colony 8  PCR  5μL + 4μL LB, 10) WELK  Colony 9 PCR  5μL + 4μL LB, 11) Negative control, 12)2-log DNA LADDER NEB 2μL + 4μL LB.
 
Fig 1.  Colony PCRs of WELK 0.8% agarose gel run at 50 min 100V. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK  Colony 1 PCR  5μL + 4μL LB, 3) WELK  Colony 2 PCR  5μL + 4μL LB, 4) WELK  Colony 3 PCR  5μL + 4μL LB, 5) WELK  Colony 4 PCR  5μL + 4μL LB, 6) WELK  Colony 5 PCR  5μL + 4μL LB, 7) WELK  Colony 6 PCR  5μL + 4μL LB, 8) WELK  Colony 7 PCR  5μL + 4μL LB, 9)WELK  Colony 8  PCR  5μL + 4μL LB, 10) WELK  Colony 9 PCR  5μL + 4μL LB, 11) Negative control, 12)2-log DNA LADDER NEB 2μL + 4μL LB.
  
Line 30: Line 32:
  
 
https://static.igem.org/mediawiki/2016/4/4f/T--TecCEM--digestion-WELKWIKI.JPG
 
https://static.igem.org/mediawiki/2016/4/4f/T--TecCEM--digestion-WELKWIKI.JPG
 +
 +
 
Fig 2. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK  Colony 7 PCR restriction (X+P) 5μL + 4μL LB, 3) WELK  Colony 9 PCR restriction (X+P)  5μL + 4μL LB, 4) WELK  Colony 1 PCR  restriction (X+P) 5μL + 4μL LB.
 
Fig 2. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK  Colony 7 PCR restriction (X+P) 5μL + 4μL LB, 3) WELK  Colony 9 PCR restriction (X+P)  5μL + 4μL LB, 4) WELK  Colony 1 PCR  restriction (X+P) 5μL + 4μL LB.
  

Latest revision as of 17:09, 25 October 2016

T4 Holin with RBS and terminator.

This biobrick contains the coding sequence for a T4 Holin (BBa_K112805) which promotes pore formation on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer. The part, designed in RFC10, was assembled with B0034 (RBS) and B0015 (double terminator). This intermediate biobrick should be cloned into a promoter-containing plasmid for its usage.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

iGEM TecCEM 2016 team achieved BBa_K1904003 documentation through Colony PCR (figure 1).

T--TecCEM--PCR-WELKWIKI.JPG


Fig 1. Colony PCRs of WELK 0.8% agarose gel run at 50 min 100V. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK Colony 1 PCR 5μL + 4μL LB, 3) WELK Colony 2 PCR 5μL + 4μL LB, 4) WELK Colony 3 PCR 5μL + 4μL LB, 5) WELK Colony 4 PCR 5μL + 4μL LB, 6) WELK Colony 5 PCR 5μL + 4μL LB, 7) WELK Colony 6 PCR 5μL + 4μL LB, 8) WELK Colony 7 PCR 5μL + 4μL LB, 9)WELK Colony 8 PCR 5μL + 4μL LB, 10) WELK Colony 9 PCR 5μL + 4μL LB, 11) Negative control, 12)2-log DNA LADDER NEB 2μL + 4μL LB.


BBa_K1904003 + PSB1A3 ligation was successful, by visualizing it through an agarose gel, since there were bands near the expected weight in several BBa_K1904003 + PSB1A3 colony samples.


To confirm and validate the presence of the insert in the aplicons, a double digestion was performed with the restriction enzymes EcoRI and PstI resulting in the expected weight (830bp), therefore confirming the presence of our biobrick.


T--TecCEM--digestion-WELKWIKI.JPG


Fig 2. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK Colony 7 PCR restriction (X+P) 5μL + 4μL LB, 3) WELK Colony 9 PCR restriction (X+P) 5μL + 4μL LB, 4) WELK Colony 1 PCR restriction (X+P) 5μL + 4μL LB.