Through the introduction of carbonic anhydrase（CA）, calcium carbonate precipitation regulator (CCPR) and the
calcium carbonate binding peptide-chintin binding domain (Cabp-Chbd) genes, Vibrio natriegens is able to fix
carbon dioxide and mineralize it.
Build
Number
Name
Description
BBa_K5397999
CARP
Coral acid-rich proteins (CARPs) not only bind Ca2+ stoichiometrically but
also precipitate aragonite in seawater at pH levels of 8.2 and 7.6 through electrostatic interactions
with protons on bicarbonate ions. Phylogenetic analysis suggests that at least one type of CARP has
evolved from a gene fusion event.
BBa_K5397987
CaBP-ChBD
CaBP-ChBD significantly promotes carbonate biomineralization on a chitin
substrate.
Test
►
We would verify expression of CA, CCPR, and Cabp-Chbd by observing fluorescence.
And we would verify the expression of sucrose by measuring its concentration.
Learn
Cycle1: Culturing
Design
►
We cultivated Vibrio natriegen using 2216E medium with varying NaCl concentrations to determine optimal
salinity. Additionally, we used M9 medium with sodium formate as the main carbon source and sucrose as a
supplement to study its growth.
Build
Tunr1
Tunr2
Tunr3
A:2216E
A:20g/L sodium formate.
A:40mM sucrose + 0mM sodium formate.
B:2216E + 1.5% NaCl
B:6g/L sucrose + 4g/L sodium formate.
B:40mM sucrose + 50mM sodium formate.
C:2216 + 3% NaCl
C:4g/L sucrose + 6g/L sodium formate.
C:40mM sucrose + 100mM sodium formate
D:2216E + 4.5% NaCl
D:Blank (M9 medium).
D:40mM sucrose + 150mM sodium formate.
Test
►
By measuring OD and modeling, we obtained the growth curve of Vibrio natriegens. The results confirmed that
V. natriegens can utilize sodium formate as the sole carbon source; however, its growth was not as robust as
in the experimental group that included sucrose as a supplementary carbon source.
Learn
►
By measuring OD and modeling, we confirmed that Vibrio natriegens can utilize sodium formate as the sole
carbon source, though growth was weaker compared to the group with sucrose supplementation. No growth
occurred without carbon sources, so a mixed carbon source group was used as a control. V. natriegens
requires adaptation in M9 medium with formate before efficiently using sodium formate alone.
Cycle2:Stain Transformation
Design
►
Literature shows two common transformation methods for Vibrio natriegens: chemical and electroporation.
Since most studies use electroporation, we adopted this method, experimenting with different voltages and
using LBV2 medium and recovery solutions. Details are provided in the protocol section.
experiment_protocol
►
We used ampicillin-resistant plates for validation. We plated 20 µL on an antibiotic-free plate and 80 µL on
an ampicillin-resistant plate to serve as a negative control. Additionally, we measured the fluorescence
intensity of mCherry to confirm the successful transformation of the strain.
Learn
►
We finally find the best transformation condition:
Voltage(V)
Concent(ms)
Electroporaton Buffer
Recover solution
1100~1200
2.5~3.0
680mM Sucrose+7mM K2HPO4
BHIv2+680mM Sucrose
Cycle3: Expression Validation
Design
►
The expression validation process includes three main components: protein extraction, chitin quality
assessment, and fluorescence detection. Protein extraction will utilize overnight-cultured strains
supplemented with chitin, while chitin quality will be assessed after adding kanamycin. Fluorescence
detection will evaluate target gene expression.
Build
►
The purpose of this table is to clearly summarize key experimental procedures for easy reference and
comparison, ensuring consistency in execution.
Procedure
Details
Protein Extraction
entrifuge at 8000 g for 10 min (4 °C), resuspend in PBS, sonicate (30% power, 2 min), centrifuge at
12000 g for 20 min (4 °C), detect protein via SDS-PAGE.
Chitin Quality Assessment
Add kanamycin (100 µg/mL), filter through 0.45 µm and 0.22 µm membranes, dry at 60 °C, weigh for
calcification rate.
Fluorescence Detection
Grow to log phase, take 200 µL samples every 10 min, measure mCherry (Ex 580 nm/Em 620 nm) and EGFP
(Ex 488 nm/Em 510 nm) over 240 min.
>
Test
►
Verify the target protein's presence using SDS-PAGE. Evaluate chitin quality based on membrane weight
measurements, and use fluorescence detection to assess mCherry and EGFP expression levels.
Learn
►
Analyze SDS-PAGE results to understand protein extraction efficiency, assess chitin quality through
calcification rates, and evaluate gene expression using fluorescence detection to optimize future protocols.
References
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