Difference between revisions of "Part:BBa K4032101"

Revision as of 06:29, 21 October 2021

lacI+RBS+α-gal-RFP+double terminator

Contents :

・The lac promoter (from BBa_R0010)

・The RBS (from BBa_B0034)

・The gene of the α-galactosidase-RFP fusion protein (from BBa_K4032005)

・The double terminator (from BBa_B0015)

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 BBa_J04450.

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

Fig. 1 The plasmid design of BBa_K40321xx

Experiments

Time course

BL21

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

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.

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; S, solubility; P, pellet.

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℃.

Sequence and Features