Part:BBa_K2094002
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
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 part BBa_K2094002. Amplification was performed via PCR. The DNA was digested with BamHI and NdeI restriction enzymes and after that 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 BL21via heat shock.
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 the expression of β-agarase. Agarolytic activity was confirmed by pit formation on the agar plates.
Assay of enzyme activity
A solution containing 4% agarose was melted and then solidified in 50 mL Erlenmeyer Flasks.
To the flasks was added:
Agarase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) [3].
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 | Sample name Figure 4 | Absorbance Value | total amount of reducing sugars |
---|---|---|---|
ß-Agarase supernatant without agar | agarase no agar | 0.209400 | 0.057727 |
in vivo positive control | agarase in vivo | 0.963100 | 0.658203 |
in vitro positive control | agarase in vitro | 0.609800 | 0.376728 |
mcherry supernatant without agar | neg. control no agar | 0.200600 | 0.050716 |
in vivo negative control | neg. control in vivo | 0.188600 | 0.041156 |
in vitro negative control | neg. control in vitro | 0.210600 | 0.058683 |
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 794
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 810
Illegal AgeI site found at 847 - 1000COMPATIBLE WITH RFC[1000]
None |