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:

Figure3: Experimental setup 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 70 rpm. E.coli BL21 with pet15b-mcherry were used as a negative control to confirm that the occurrence of reducing sugars is due to the β-Agarases and not to other metabolic pathways. By using bacteria having the same Plasmid but with another insert, a possible influence of the pet-15b vector can also be ruled out Created with BioRender.com

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

3,5-dinitrosalicylic acid method (Miller 1959).jpeg

Briefly, 1.5 mL of sample solution was mixed with 0.5 mL of DNS reagent, the reaction was heated in boiling water for 5 min and then placed on ice for 5 min. Absorbance was measured at a wavelength of 540 nm, a standard curve of D-Galactose was used to determine the total amount of reducing sugars.

Samples were measured as follows:

Sample	Absorbance Value	total amount of reducing sugars
ß-Agarase supernatant without agar	e	f
in vivo positive control	h	i
in vitro positive control	z	x
mcherry supernatant without agar	y	b
in vivo negative control	n	m
in vitro negative control	u	o

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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Unknown
12
INCOMPATIBLE WITH RFC[12]
Unknown
21
INCOMPATIBLE WITH RFC[21]
Unknown
23
INCOMPATIBLE WITH RFC[23]
Unknown
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 810
Illegal AgeI site found at 847
1000
COMPATIBLE WITH RFC[1000]