Part:BBa_K4032101
lacI+RBS+α-gal-RFP+double terminator
Contents :
・The lac promoter (https://parts.igem.org/Part:BBa_R0010)
・The RBS (https://parts.igem.org/Part:BBa_B0034)
・The gene of the α-galactosidase-RFP fusion protein.
・The double terminator (https://parts.igem.org/Part:BBa_B0015)
Usage and Biology
This enzyme catalyzes the hydrolysis of terminal, non-reducing alpha-D-galactose residues in alpha-D-galactosides, including galactose oligosaccharides, galactomannans and galactolipids.
Red fluorescence of RFP is observed under the fluorescence microscope and natural light.
Design
This part expresses the α-galactosidase-RFP fusion protein.
α-galactosidase is from E.coli K-12 MG1655 and RFP is from BBa_J04450 (https://parts.igem.org/Part:BBa_J04450).
We removed the stop codon of alpha-galactosidase and the start codon of RFP and connected the ends.
Fig. 1 The plasmid design of BBa_K40321xx
Experiments
Time course
BL21
Fig. 2 The growth of E.coli (BL21(DE3)) expressing of alpha-galactosidase
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 of alpha-galactosidase
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 Escherichia coli at 37 ° C. 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 Escherichia coli was cultured overnight at 25 ° C. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2131
Illegal AgeI site found at 2243 - 1000COMPATIBLE WITH RFC[1000]
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