Difference between revisions of "Part:BBa K4180005"

Latest revision as of 03:33, 12 October 2022

pGal1,10-SNF1

This pGal1,10-SNF1 is a composite biobrick. pGal1, 10-SPT5-Streptavidin Binding Protein(SBP) plasmid, containing 2u origin of replication (ORI) for yeast to start DNA replication, and ORI for bacteria DNA replication, was sponsored by Dr. Tien-Hsien Chang, at Genomics Research Center, Academia Sinica, in Taipei Taiwan. This plasmid can be transformed into bacteria and yeast, which was beneficial for our team to finish making biobricks in bacteria and perform the functional assay in yeast. Our team made 5 different basic parts (one promoter and 4 coding regions) and 4 different composite parts by inserting those 4 basic parts (BBa_K4180000, BBa_K4180002, BBa_K4180003, and BBa_K4180004) to replace the original SPT5 gene.

Galactose promoter can be induced in the presence of galactose and express SNF1(FL) ; SNF1 in yeast is homology of AMPKα in human, which is a major metabolism sensor protein of the whole metabolic pathways, increasing catabolic process, such as triggering fatty acid catabolism for more ATP, and reducing anabolic process, such as protein and fatty acid synthesis to reduce energy consumed.

Citation: 1. Herzig, S. and R. J. Shaw (2018). "AMPK: guardian of metabolism and mitochondrial homeostasis." Nat Rev Mol Cell Biol 19(2): 121-135.
2. Yang, T T et al. “Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.” The Journal of biological chemistry vol. 273,14 (1998): 8212-6. doi:10.1074/jbc.273.14.8212

Sequence and Features

<BBa_K4180005 in BY4741>

BBa_K4180005 composite site was SNF1 full-length coding region cloned downstream of the Gal1, 10 promoter. BBa_K4180005 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 3-fold at 17, and 24 hr, and 5-fold at 41 hr, which didn’t show if the BBa_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in BBa_K4180008 control, since BBa_K4180005 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa_K4180008. Compared to the endogenous SNF1 inBY4741, the SNF1 induction in BBa_K4180005 composite site was suppressed in the presence of galactose at 24hr.

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1192
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 286
1000
COMPATIBLE WITH RFC[1000]

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 This pGal1,10-SNF1 is a composite biobrick. pGal1, 10-SPT5-Streptavidin Binding Protein(SBP) plasmid, containing 2u origin of replication (ORI) for yeast to start DNA replication, and ORI for bacteria DNA replication, was sponsored by Dr. Tien-Hsien Chang, at Genomics Research Center, Academia Sinica, in Taipei Taiwan. This plasmid can be transformed into bacteria and yeast, which was beneficial for our team to finish making biobricks in bacteria and perform the functional assay in yeast. Our team made 5 different basic parts (one promoter and 4 coding regions) and 4 different composite parts by inserting those 4 basic parts (BBa_K4180000, BBa_K4180002, BBa_K4180003, and BBa_K4180004) to replace the original SPT5 gene.
+Galactose promoter can be induced in the presence of galactose and express SNF1(FL) ; SNF1 in yeast is homology of AMPKα in human, which is a major metabolism sensor protein of the whole metabolic pathways, increasing catabolic process, such as triggering fatty acid catabolism for more ATP, and reducing anabolic process, such as protein and fatty acid synthesis to reduce energy consumed.<br><br>
+Citation:  &emsp;1. Herzig, S. and R. J. Shaw (2018). "AMPK: guardian of metabolism and mitochondrial homeostasis." Nat Rev Mol Cell Biol 19(2): 121-135.<br>
+&emsp;&emsp;&emsp;&emsp;&emsp;2. Yang, T T et al. “Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.” The Journal of biological chemistry vol. 273,14 (1998): 8212-6. doi:10.1074/jbc.273.14.8212<br>
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 [[File:BBa_K4180005.jpeg |250px||right|]]
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+<!-- --><BBa_K4180005 in BY4741>
-BBa_K4180005 in BY4741 use SNF1-1 primers to do qPCR
 [[File: BBa K4180005 in BY4741.jpeg|500px|]]
-Fig5:BBa_K4180005 composite site was SNF1 full length coding region cloned downstream of the Gal1, 10 promoter. BBa_K4180005 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 3-fold at 17, and 24 hr, and 5-fold at 41 hr, which didn’t show if the BBa_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in  BBa_K4180008 control, since BBa_K4180005 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in  BBa_K4180008 on fig 3. Compared to the fig 1, endogenous SNF1 inBY4741, the SNF1 induction in BBa_K4180005 composite site was suppressed in the presence of galactose at 24hr.
+BBa_K4180005 composite site was SNF1 full-length coding region cloned downstream of the Gal1, 10 promoter. BBa_K4180005 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 3-fold at 17, and 24 hr, and 5-fold at 41 hr, which didn’t show if the BBa_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in  BBa_K4180008 control, since BBa_K4180005 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in  BBa_K4180008. Compared to the endogenous SNF1 inBY4741, the SNF1 induction in BBa_K4180005 composite site was suppressed in the presence of galactose at 24hr.
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