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Team:Hong Kong HKUST/experiment/exp2
From 2013.igem.org
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| - | <br><br><br><div id="slide"><center><h3 class="title"> | + | <br><br><br><div id="slide"><center><h3 class="title">Fatty Acid Sensing Mechanism</h3></center><br> |
<h1>BBa_J176171</h1> | <h1>BBa_J176171</h1> | ||
<p id="yo">BBa_ J176171 was planned to be used as a mammalian vector backbone for the promoters. After running PCR (Polymerase Chain Reaction) to amplify the FABP1 promoter and ligated it with GFP and the backbone, we did mutagenesis for the vector. Based on the gel photo, we concluded that the vector was easy to get degraded. Finally we figured out that BBa-J176171 was heat unstable and changed our plan.</p><br> | <p id="yo">BBa_ J176171 was planned to be used as a mammalian vector backbone for the promoters. After running PCR (Polymerase Chain Reaction) to amplify the FABP1 promoter and ligated it with GFP and the backbone, we did mutagenesis for the vector. Based on the gel photo, we concluded that the vector was easy to get degraded. Finally we figured out that BBa-J176171 was heat unstable and changed our plan.</p><br> | ||
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| + | <h1>Liver Fatty Acid Binding Protein 1 (FABP1) Promoter </h1> | ||
| + | <p id="yo">This promoter was cloned from human genomic DNA (gDNA) (see <a href="https://static.igem.org/mediawiki/2013/f/f1/14._Isolation_of_gDNA_for_mammalian_cells.pdf">gDNA extraction</a>and <a href="https://static.igem.org/mediawiki/2013/0/07/8._PCR.pdf">PCR protocols</a>) with engineered RFC10 prefix and suffix for BioBrick submission. The promoter was then ligated with enhanced green fluorescence protein (eGFP) from pEGFP-N1 (Addgene), and cloned into BBa_J176171 mammalian backbone. The promoter and eGFP were successfully cloned into BBa_J176171 by digestion and ligation. </p> | ||
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| + | The confirmed construct was transfected into two mammalian cells, HEK293FT and HepG2 cell lines. GFP signal of the construct was compared with pEGF-N1 plasmid that contains constitutive CMV promoter. However, no GFP signal could be detected. | ||
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| + | Since FABP1 promoter contained illegal restriction sites for BioBrick submission, we conducted multi site-directed mutagenesis to elimitate EcoRI and PstI from the coding sequence. (see Mutagenesis protocol 1,2 and 3 ). | ||
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| + | The FABP1 promoter was to be characterized by over-expression of eGFP reporter in the presence of high fatty acid concentration in the medium1. However, no eGFP signal could be detected. | ||
| + | |||
| + | After several attempts of site-mutagenesis of promoter in FABP1 – eGFP – BBa_J176171 construct, we found that the plasmid degrades at a high temperature. We tested heat sensitivity of the plasmid BBa_J176171 and found that the plasmid degrades at denaturation step (95 °C) of polymerase chain reaction. Then, we took an alternative experiment by cloning FABP1 promoter into pBlueScript KS(+). Due to time constrain, two mutagenesis attempts were taken but both of them were not successful. | ||
| + | </p><br> | ||
</div> | </div> | ||
Revision as of 18:49, 27 September 2013
Fatty Acid Sensing Mechanism
BBa_J176171
BBa_ J176171 was planned to be used as a mammalian vector backbone for the promoters. After running PCR (Polymerase Chain Reaction) to amplify the FABP1 promoter and ligated it with GFP and the backbone, we did mutagenesis for the vector. Based on the gel photo, we concluded that the vector was easy to get degraded. Finally we figured out that BBa-J176171 was heat unstable and changed our plan.
Liver Fatty Acid Binding Protein 1 (FABP1) Promoter
This promoter was cloned from human genomic DNA (gDNA) (see gDNA extractionand PCR protocols) with engineered RFC10 prefix and suffix for BioBrick submission. The promoter was then ligated with enhanced green fluorescence protein (eGFP) from pEGFP-N1 (Addgene), and cloned into BBa_J176171 mammalian backbone. The promoter and eGFP were successfully cloned into BBa_J176171 by digestion and ligation.
The confirmed construct was transfected into two mammalian cells, HEK293FT and HepG2 cell lines. GFP signal of the construct was compared with pEGF-N1 plasmid that contains constitutive CMV promoter. However, no GFP signal could be detected. Since FABP1 promoter contained illegal restriction sites for BioBrick submission, we conducted multi site-directed mutagenesis to elimitate EcoRI and PstI from the coding sequence. (see Mutagenesis protocol 1,2 and 3 ). The FABP1 promoter was to be characterized by over-expression of eGFP reporter in the presence of high fatty acid concentration in the medium1. However, no eGFP signal could be detected. After several attempts of site-mutagenesis of promoter in FABP1 – eGFP – BBa_J176171 construct, we found that the plasmid degrades at a high temperature. We tested heat sensitivity of the plasmid BBa_J176171 and found that the plasmid degrades at denaturation step (95 °C) of polymerase chain reaction. Then, we took an alternative experiment by cloning FABP1 promoter into pBlueScript KS(+). Due to time constrain, two mutagenesis attempts were taken but both of them were not successful.
