Difference between revisions of "Team:Waterloo/InDepthDesign/PromoterSelection"

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It was necessary for us to choose promoters that expressed the constructs we were cloning into yeast cells. We wanted to characterize promoter expression with GFP before we decided which promoter to use in our system with the CFP-Hsp104 construct. Furthermore, we needed to induce a [PSI+] state by overexpression of Sup35 in the cell by inserting another copy of Sup35 into the system via a plasmid. Overall, we had two plasmids that we wanted to easily induce at any time.
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We chose to characterize four main promoters that are commonly found in yeast: Gal1, Adh1, Met25, and Cup1. We cloned all of these into the pXP218 plasmid and then transformed them into yeast to test their strength with and without the presence of inducers. All of the promoters we picked are inducible except for Adh1, which acts constitutively. We made the promoter constructs by the traditional restriction digest cloning method after order the synthesized promoters with a GFP fluorescent marker and doing PCR amplification with appropriate primers.
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Specifically, The promoters were added into the pXP218 plasmid between the restriction enzymes SphI and SalI, which are near the multiple cloning site in the plasmid.The criteria required for the plasmid were the following: there are no T7 promoter regions near the promoter insertion site, so that T7 does not give a false positive by increasing expression of the promoter being tested, it had to be able to replicate in yeast, be able to be constructed in Escherichia coli,  have a CEN/ARS or a 2 micron origin of replication, and have a bacterial origin of replication. Having a selective marker in the plasmid was also important an important factor so that we were able to select for transformants into yeast; some markers include LEU2, URA3, HIS3.
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Vector Backbone: pUC18
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Backbone Size (bp): 6226
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Vector Type: Yeast Expression
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Selectable Markers: URA3
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Bacterial Resistance(s): Ampicillin
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Growth Temperature: 37 ºC
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Growth Strain(s): DH5alpha
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Copy Number: High
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Revision as of 03:35, 12 October 2016

Title

It was necessary for us to choose promoters that expressed the constructs we were cloning into yeast cells. We wanted to characterize promoter expression with GFP before we decided which promoter to use in our system with the CFP-Hsp104 construct. Furthermore, we needed to induce a [PSI+] state by overexpression of Sup35 in the cell by inserting another copy of Sup35 into the system via a plasmid. Overall, we had two plasmids that we wanted to easily induce at any time.

We chose to characterize four main promoters that are commonly found in yeast: Gal1, Adh1, Met25, and Cup1. We cloned all of these into the pXP218 plasmid and then transformed them into yeast to test their strength with and without the presence of inducers. All of the promoters we picked are inducible except for Adh1, which acts constitutively. We made the promoter constructs by the traditional restriction digest cloning method after order the synthesized promoters with a GFP fluorescent marker and doing PCR amplification with appropriate primers.

Specifically, The promoters were added into the pXP218 plasmid between the restriction enzymes SphI and SalI, which are near the multiple cloning site in the plasmid.The criteria required for the plasmid were the following: there are no T7 promoter regions near the promoter insertion site, so that T7 does not give a false positive by increasing expression of the promoter being tested, it had to be able to replicate in yeast, be able to be constructed in Escherichia coli, have a CEN/ARS or a 2 micron origin of replication, and have a bacterial origin of replication. Having a selective marker in the plasmid was also important an important factor so that we were able to select for transformants into yeast; some markers include LEU2, URA3, HIS3.

  • Vector Backbone: pUC18 Backbone Size (bp): 6226 Vector Type: Yeast Expression Selectable Markers: URA3 Bacterial Resistance(s): Ampicillin Growth Temperature: 37 ºC Growth Strain(s): DH5alpha Copy Number: High

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    Content