IGEM:Caltech/2008/Project/Lactose intolerance: Difference between revisions

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Plasmid #1 - A constitutive promoter with <i>LacZ</i> and <i>LacY</i>.<br>
Plasmid #1 - A constitutive promoter with <i>LacZ</i> and <i>LacY</i>.<br>
Plasmid #2 - A lactose inducible promoter with lysozyme and holin.<br>
Plasmid #2 - A lactose inducible promoter with lysozyme and holin.<br>
The general idea is to overexpress LacY (Lactose transporter) with LacZ (Beta-Galactosidase). When lactose is present, the cell will uptake lactose and will flip on the Lac promoter repressed by lacI when enough lactose is present. We then express lysozyme and holin which will lyse the cell releasing beta-gal cleaving lactose into glucose and galactose. Hopefully, glucose and galactose will be taken up by the host in the colon.
The general idea is to overexpress LacY (Lactose transporter) with LacZ (Beta-Galactosidase). When lactose is present, the cell will uptake lactose and will flip on the Lac promoter repressed by lacI when enough lactose is present. We then express lysozyme and holin which will lyse the cell releasing beta-gal cleaving lactose into glucose and galactose. Glucose and galactose will then be taken up by the host in the colon.<br>
==[[/Lactose Outline|Lactose Outline]]==


==Lactose Outline==
<b>6.19.08</b><br>
We have decided to use the I732017 LacZ (1009/11H) because we could not transform I732005 LacZ (1018/6F).<br>
To double check, we will plate our transformed I732005 LacZ with 100μL onto a kan plate, and the rest on an Amp plate.<br>
We ordered primers for LacY, and we will need to PCR out LacY. We then need to run site directed mutagenesis on LacY by added 2H to prevent EIIA(GLUC) binding.<br>
For our constitutive promoter, we will use various strengths to test our beta-gal concentrations.<br>
Low expression - J23113<br>
Medium expression - J23106<br>
High expression - J23100<br>
We need to cut out mRFP, which is originally in the promoter plasmid family of J23100, by doing a digest with SpeI and PstI. We will paste in LacZ and LacY, and add a double TT.<br>
<b>6.20.08</b><br>
Colonies grew from I732017 LacZ transformation. They will be cultured Sunday afternoon.<br>
LacY, LacY +H, and LacY +2H has been inputted into the registry of parts. Primers for LacY site directed mutagenesis were designed.<br>When primers for LacY arrive, need to PCR LacY out of E. coli genomic DNA.


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Latest revision as of 15:52, 15 August 2008

iGEM 2008

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Lactose Summary

We want to make two plasmids:
Plasmid #1 - A constitutive promoter with LacZ and LacY.
Plasmid #2 - A lactose inducible promoter with lysozyme and holin.
The general idea is to overexpress LacY (Lactose transporter) with LacZ (Beta-Galactosidase). When lactose is present, the cell will uptake lactose and will flip on the Lac promoter repressed by lacI when enough lactose is present. We then express lysozyme and holin which will lyse the cell releasing beta-gal cleaving lactose into glucose and galactose. Glucose and galactose will then be taken up by the host in the colon.

Lactose Outline

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