Difference between revisions of "Part:BBa K1144001:Experience"

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<I>HUST-China 2015</I>
 
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<I>HUST-China 2015</I>
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The CRY2/CIB1 interaction is entirely genetically encoded and does not require addition of any exogenous cofactors. The binding naturally reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark.
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This fusion protein is for use in a yeast-two-hybrid system, and a Gal4 DNA binding domain fused to its C terminus.
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To regulate DNA transcription by blue light, the system is based on a two-hybrid interaction in which a light-mediated protein interaction brings together two halves (a binding domain and an activation domain) of a split transcription factor. If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIB1 and halt Gal4-dependent transcription.
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[[File:yeast-two-hybrid.png|600px|thumb|center|The principle of blue-light-control system based on yeast-two-hybrid.]]
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<h1>'''Characterization'''</h1>
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From addgene, we received a plasmid pRMH120 that containing both Gal4BD-CRY2 and Gal4AD-CIB1 fusions on a p414TEF backbone. These two fusions are under the control of constitutive promoter P<sub>TEF1</sub> and P<sub>ADH1</sub> respectively. Since promoter'''UAS-PGal1''' and downstream gene β-galactosidase exists in yeast Y187 originally, we can validate the light-control system by testing the activity of β-galactosidase. Thus, we use Saccharomyces cerevisiae Y187 as chassis to test the light-control system.
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[[File:HUST-China_2015_results_1.jpg|600px|thumb|center|Figure3: β-galactosidase activity of CRY2-CIB1 system tested in darkness or light. The control group was wildtype Y187. (Error bars represent sample standard error, n = 4).]]
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Experiments were carried out three times with similar results to show. We observed that pRMH120 transformed cells incubated in white light (18W) gave distinguishable activation from pRMH120 tansformed cells incubated in total darkness, which means that GalBD-CRY2 coupled well with GalAD-CIB1 for light-inducible protein expression. Considering AD and BD could bind randomly, the result that the strains with pRMH120 in darkness was also activated a bit than the control wildtype yeast is reasonable.
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Revision as of 21:00, 17 September 2015


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1144001

User Reviews

UNIQ4f9195b91c2cfd64-partinfo-00000000-QINU UNIQ4f9195b91c2cfd64-partinfo-00000001-QINU

••••

HUST-China 2015

The CRY2/CIB1 interaction is entirely genetically encoded and does not require addition of any exogenous cofactors. The binding naturally reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in a yeast-two-hybrid system, and a Gal4 DNA binding domain fused to its C terminus. To regulate DNA transcription by blue light, the system is based on a two-hybrid interaction in which a light-mediated protein interaction brings together two halves (a binding domain and an activation domain) of a split transcription factor. If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIB1 and halt Gal4-dependent transcription.

The principle of blue-light-control system based on yeast-two-hybrid.


Characterization

From addgene, we received a plasmid pRMH120 that containing both Gal4BD-CRY2 and Gal4AD-CIB1 fusions on a p414TEF backbone. These two fusions are under the control of constitutive promoter PTEF1 and PADH1 respectively. Since promoterUAS-PGal1 and downstream gene β-galactosidase exists in yeast Y187 originally, we can validate the light-control system by testing the activity of β-galactosidase. Thus, we use Saccharomyces cerevisiae Y187 as chassis to test the light-control system.


Figure3: β-galactosidase activity of CRY2-CIB1 system tested in darkness or light. The control group was wildtype Y187. (Error bars represent sample standard error, n = 4).


Experiments were carried out three times with similar results to show. We observed that pRMH120 transformed cells incubated in white light (18W) gave distinguishable activation from pRMH120 tansformed cells incubated in total darkness, which means that GalBD-CRY2 coupled well with GalAD-CIB1 for light-inducible protein expression. Considering AD and BD could bind randomly, the result that the strains with pRMH120 in darkness was also activated a bit than the control wildtype yeast is reasonable.

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