View source for Part:BBa K4032101


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<partinfo>BBa_K4032101 short</partinfo>

Contents :

・The lac promoter (from <partinfo>BBa_R0010</partinfo>)

・The RBS (from <partinfo>BBa_B0034</partinfo>)

・The gene of the α-galactosidase-RFP fusion protein (from <partinfo>BBa_K4032005</partinfo>)

・The double terminator (from <partinfo>BBa_B0015</partinfo>)



===Usage and Biology===

This enzyme catalyzes the hydrolysis of terminal, non-reducing α-D-galactose residues in α-D-galactosides, including galactose oligosaccharides, galactomannans and galactolipids.

Red fluorescence of RFP is observed not only with a fluorescence microscope and under the natural light source.


===Design===

This part expresses the α-galactosidase-RFP fusion protein.

α-galactosidase is from ''E. coli'' and RFP is from <partinfo>BBa_J04450</partinfo>.

We removed the stop codon of the α-galactosidase gene and the start codon of the RFP gene and connected the two ends.



https://2021.igem.org/wiki/images/thumb/9/96/T--Gunma--%CE%B1-gal-RFP-design-2.png/800px-T--Gunma--%CE%B1-gal-RFP-design-2.png


Fig. 1 The plasmid design of BBa_K4032101



==Experiments==

===Time course===
BL21(DE3)


https://2021.igem.org/wiki/images/c/cb/T--Gunma--%CE%B1-galactosidase-RFP-timecourse-BL21.png


Fig. 2 The growth of ''E. coli'' (BL21(DE3)) expressing α-galactosidase-RFP fusion protein

Pre-culture: 37 ℃,16 h (130 rpm)

Culture: 37 ℃ (130rpm)

･Measuring OD600 every 4 hours



DH5α


https://2021.igem.org/wiki/images/4/4c/T--Gunma--%CE%B1-galactosidase-RFP-timecourse-dh5%CE%B1.png


Fig. 3  The growth of ''E. coli'' (DH5α)) expressing α-galactosidase-RFP fusion protein 

Pre-culture: 37 ℃,16 h (130 rpm)

Culture: 37 ℃ (130rpm)

･Measuring OD600 every 4 hours



===SDS-PAGE===

In order to investigate the expression of a plasmid in which α-galactosidase derived from ''Escherichia coli'' and RFP derived from coral were fused, DH5α-transformed bacteria were cultured in an LB medium containing chloramphenicol. After culturing ''E. coli'' at 37 ℃. and 130 rpm for 16 hours, the cells were inoculated into a new medium and liquid-cultured until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and ''E. coli'' was cultured overnight at 25 ℃. and 130 rpm for 10 hours.

Cultured cells were sonicated with sonication buffer (phosphate buffer (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.


[[image:T--Gunma--α-galactosidase-RFP-SDS-PAGE.png|400px|thumb|left]]





















Fig. 4  SDS-PAGE gel for quantification of α-galactosidase-RFP. M, molecular mass markers; WT, wild type; α-Gal-RFP, α-galactosidase-RFP from ''E. coli'' and coral; P, pellet S, soluble.



===Test===

To examine whether α-gal-RFP has an enzymatic activity in vitro, we measured degradation of PNPG that was used as a substrate of α-galactosidase. PNPG (4-Nitrophenyl-β-D- glucopyranoside) is a chromogenic β-D-glucosidase substrate, producing a yellow solution on cleavage.


These results confirmed that a partially purified fraction of α-gal-RFP exhibited an α-gal activity level of α-gal. It is noteworthy that the activity was observed even at 65 ℃, although with about 40% activity compared to that at 37 ℃. 

https://2021.igem.org/wiki/images/thumb/1/18/T--Gunma--%CE%B1-galactosidase-RFP-activitytest.png/800px-T--Gunma--%CE%B1-galactosidase-RFP-activitytest.png




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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K4032101 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K4032101 parameters</partinfo>
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