Revision as of 09:40, 7 October 2021

beta-agarase YM01-3

This enzyme hydrolyzes the β-1,4-glycosidic linkages of agarose.

Contribution

• Group: iGEM Team Heidelberg 2021
• Author: Franziska Giessler
• Summary: The Part BBa_K2094002 was used for our project and further characterized by enzyme activity measurements.

Background

Figure1: Enzyme activity of the β-agarases

Agar is often used in the food industry as a thickening agent or as a vegan alternative to gelatin. It is a complex polysaccharide consisting of alternating 3-O-linked β-D-galactopyranose and 4-O-linked α-L-galactopyranose. Agar cannot be degraded by most microorganisms, but there are some bacteria that metabolize agar as a carbon and energy source. They are mainly found in marine environments, where food resources are limited and agar is abundant in the form of the cell wall of some algae [1],[2].

The idea is to use the ability of agar degradation as a selection advantage for specific bacteria in order to overcome the established antibiotic selection used in the laboratory.

One of the enzymes present in agarolytic bacteria is the β-Agarase that hydrolyzes the β-(1,4) glycosidic bonds (see Figure 1).

Experiments and Results

Cloning

The DNA was synthesized using the sequence from the part (Ba_K2094002). Amplification was performed via PCR. The DNA was digested with BamHI and NdeI restriction enzymes and then ligated with a T4 ligase into a pet15b backbone. This construct includes a T7 promoter, lac operator and an ampicillin resistance. The construct was transformed into competent E.coli BL21.

Culturing

Transformed E.coli Bl21 were cultured on LB agar plates with carbenicillin for antibiotic selection and isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce expression. Agarolytic activity was confirmed by pit formation on the agar plates.

Assay of enzyme activity

A solution containing 4% agarose was meted and then solidified in 50 mL Erlenmeyer Flask. To the flasks was was added:

a) in vivo positive control: 5mL 1:10 dilution of an overnight culture of E.coli BL21 with pet15b_β-Agarase Plasmid b) in vitro positive control: 5 mL supernatant of overnight culture E.coli BL21 with pet15b_β-Agarase Plasmid c) in vivo negative control: 5mL 1:10 dilution of overnight culture E.coli BL21 with pet15b_mcherry Plasmid d) in vitro positive control: 5 mL supernatant of overnight culture E.coli BL21 with pet15b_mcherry Plasmid

To the overnight cultures as well as to the in vivo experiments, carbenicillin was added for selection and IPTG was added to induce expression.

Samples were incubated for 12h at 37 C 70rpm.

Agarase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) [3].

Reference

[1]Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Agar degradation by microorganisms and agar-degrading enzymes. Applied microbiology and biotechnology, 94(4), 917–930. https://doi.org/10.1007/s00253-012-4023-2

[2]Su, Q., Jin, T., Yu, Y., Yang, M., Mou, H., & Li, L. (2017). Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber. AMB Express, 7(1), 220. https://doi.org/10.1186/s13568-017-0525-8

[3]G. L. Miller. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry. Vol. 31(3):426-428. DOI: 10.1021/ac60147a030

Sequence and Features