Composite

Part:BBa_K4032101

Designed by: Tsugumi Ohzeki   Group: iGEM21_Gunma   (2021-09-30)
Revision as of 06:43, 21 October 2021 by Tsubasa O (Talk | contribs)


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.


800px-T--Gunma--%CE%B1-gal-RFP-design-2.png


Fig. 1 The plasmid design of BBa_K40321xx


Experiments

Time course

BL21(DE3)


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


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.


560px-T--Gunma--%CE%B1-galactosidase-RFP-SDS-PAGE.png


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℃.

800px-T--Gunma--%CE%B1-galactosidase-RFP-activitytest.png



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2131
    Illegal AgeI site found at 2243
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None