Difference between revisions of "Part:BBa K1904003"
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<partinfo>BBa_K1904003 short</partinfo> | <partinfo>BBa_K1904003 short</partinfo> | ||
| − | This biobrick | + | 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. |
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K1904003 parameters</partinfo> | <partinfo>BBa_K1904003 parameters</partinfo> | ||
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| + | iGEM TecCEM 2016 team achieved BBa_K1904003 documentation through Colony PCR (figure 1). | ||
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| + | https://static.igem.org/mediawiki/2016/d/da/T--TecCEM--PCR-WELKWIKI.JPG | ||
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| + | 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. | ||
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| + | 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. | ||
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| + | 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. | ||
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| + | https://static.igem.org/mediawiki/2016/4/4f/T--TecCEM--digestion-WELKWIKI.JPG | ||
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| + | 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. | ||
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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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
iGEM TecCEM 2016 team achieved BBa_K1904003 documentation through Colony PCR (figure 1).
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.
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.