Difference between revisions of "Part:BBa K4665005"

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A Bradford assay was carried out in order to determine protein concentration for the SazCA-INPN mineralisation module. A low working range (1-25µg/mL) microplate protocol was followed according to NanoDrop values. Working volumes of standard/sample was 150µL, with an equal volume of Bradford Protein Assay Reagent (ThermoFisher). Absorbance was measured at 595 using a microplate reader.  
 
A Bradford assay was carried out in order to determine protein concentration for the SazCA-INPN mineralisation module. A low working range (1-25µg/mL) microplate protocol was followed according to NanoDrop values. Working volumes of standard/sample was 150µL, with an equal volume of Bradford Protein Assay Reagent (ThermoFisher). Absorbance was measured at 595 using a microplate reader.  
 
Average protein concentration was determined to be 68.34 µg/mL (0.5 mM ZnSO4 + 0.5 mM IPTG).  
 
Average protein concentration was determined to be 68.34 µg/mL (0.5 mM ZnSO4 + 0.5 mM IPTG).  
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<b>In Vitro Mineralization:</b>
 
<b>In Vitro Mineralization:</b>

Revision as of 18:15, 8 October 2023

SazCA-INPN Membrane Display Module

Usage and Biology

Biomineralization is the process by which living organisms synthesise minerals (Dhami et al., 2013). Microbial calcium carbonate production can proceed through two main metabolic pathways, using urease or carbonic anhydrase (CA) as the catalysts of the reaction (Chaparro-Acuña et al., 2019). However, synthesis through urea hydrolysis produces toxic byproducts which is not observed in the CA catalyzed pathway.

SazCA, derived from the thermophilic bacterium Sulfurihydrogenibium azorense, is the fastest known carbonic anhydrase to date, with an approximate kcat/KM value of 3.5 × 108 M−1 s−1 (De Simone et al, 2015; De Luca et al., 2013). SazCA facilitates the hydration of carbon dioxide to bicarbonate and protons, creating alkaline conditions that aid the formation of calcium carbonate crystals on the extracellular matrix (EPS) of bacterial cells (Fig. 1) (Anbu, et al., 2016).

Image 1
To enhance enzymatic efficiency, this composite part expresses the SazCA enzyme as a fusion protein on the cell surface of E. coli. This approach bypasses cellular limitations and directly exposes the enzyme to extracellular CO2, increasing calcium carbonate production on limestone surfaces. This component is based on the findings of Zhu et al. (2022), wherein a membrane fusion protein was designed to showcase SazCA on the surface of E. coli cells. This is achieved by linking the E. coli codon-optimized SazCA enzyme (BBa_K4665120) to the integral membrane protein INPN (BBa_K4665001) using a flexible GGGGS linker (BBa_K4665175).


This composite part consists of three basic parts:

1) Ice nucleation protein N-terminal (INPN): This is the N-terminal of ice nucleation protein which will be embedded into the E. coli cell membrane. The sequence coding for the INPN is preceded by a pelB leader sequence as its expression promotes the secretion of the protein via the Sec pathway whilst avoiding hydrolysis by cytoplasmic proteases that might lower the quantity of proteins on the cell’s surface (Mergulhao et al., 2005). By attaching the pelB signal peptide in front of the INP protein, the fusion protein will be directed towards the bacterial periplasm where it will be anchored in the cell membrane (Singh et al., 2013). The INPN sequence is followed by two front-end sub-repeat sequences important for the stability of the fusion protein (Zhu et al., 2022).

2) GGGGS linker: The GGGGS flexible linker is composed of a sequence of 4 glycine repeats followed by a serine amino acid. This flexible linker is used to connect the N-terminal of the INP to the carbonic anhydrase which creates an elongated fusion mode that allows for optimal carbonic anhydrase stability (Hartmann et al., 2022; Zhu et al., 2022).

3) SazCA: This sequence codes for the carbonic anhydrase derived from Sulfurihydrogenibium azorense (SazCA). This sequence has been codon optimised for E. coli. The SazCA coding sequence is followed by a His-tag which facilitates the purification and detection of the fusion protein.
Image 1 Figure taken from Zhu et al., (2021).

Zhu et al. (2022) were able to show that the surface display of the INP-SazCA fusion protein significantly elevates the enzyme’s stability, optimising whole-cell activity at 25°C and pH 9, retaining minimal metal inhibition.

Characterisation

Expression:

After successful transformation into BL21 DE3 E. coli, the expression of our recombinant protein was tested. The construct is preceded by the T7 promoter, therefore expression of recombinant protein can be induced through addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). Liquid overnight cultures of the transformed E. coli were induced with IPTG and samples were taken at different incubation times. Membrane protein extraction was performed by incubation with NPI-10 buffer followed by sonication. SDS PAGE and Western Blot was performed with antibodies that bind to the 6X-His tag attached at the end of the fusion protein. Previous iterations of the Western Blot revealed the problem of a leaky T7 promoter in our recombinant plasmid, leading to protein expression in absence of IPTG. Previous research has shown that addition of glucose to BL21 DE3 bacterial culture can reduce the background expression of the uninduced gene (Pan & Malcolm, 2000). In our Western Blot we tested various controls, the transformed BL21 DE3 cultures induced with IPTG at different time intervals and the transformed BL21 DE3 cultures that were grown overnight in the presence of glucose. Prior to IPTG induction, the glucose-containing medium was removed from the 0.5% and 1% glucose samples and they were resuspended in normal LB medium to prevent reduced expression of SazCA during IPTG induction.


Gel 1: Image 1
Protein ladder
1. Not induced GG3B
2. Negative control BL21 DE3
3. Not induced 0.5% glucose
4. Not induced 1% glucose
5. IPTG Induced GG3B 1hr
6. Induced GG3B 0.5% 1hr
7. Induced GG3B 1% hr
Gel 2: Image 1
Protein ladder -
8. IPTG Induced GG3B 2hr
9. Induced GG3B 0.5% 2hr
10. Induced GG3B 1% 2hr


The absence of a band in the BL21 DE3 control confirms that the observed bands in uninduced samples are due to a leaky T7 promoter and not due to the presence of native histidine-rich proteins expressed in BL21 DE3 E. coli . The results show that the addition of 0.5% or 1% glucose in the growth medium leads to reduced expression of the recombinant protein in the uninduced samples. However, despite the removal of the glucose-containing medium, the samples that contained the glucose showed reduced expression during IPTG induction. Our recombinant protein was most abundant in the transformed BL21 DE3 culture that had been induced with IPTG for 2 hours.

Protein Quantification

Cells from liquid culture (LB + Kanamycin + 0.5 mM ZnSO4 + 0.5 mM IPTG) were sonicated to lyse the cell membrane. Prior to the procedure, cells were centrifuged and shocked at -80℃ for 1h. Pellets were resuspended in 3x volume of NPI-10 buffer (50mM NaH2PO4, 300mM NaCl, 10mM Imidazole) and left on ice for 30 minutes. Samples were sonicated on ice using a 40 kHz pulse applied for 30s, followed by a 2 min pause. This sonication step was repeated 10 times. Given the fact that the construct is a protein embedded protein, a cell fractionation step should be followed by sonication. Unfortunately, this step was not performed due to lack of proper available equipment (ultracentrifuge). Protein extraction was performed using affinity chromatography with HisPur™ Ni-NTA Resin (ThermoFisher), optimising yield through the addition of Triton-100X to the protein buffers to reduce nonspecific interactions with untagged proteins. Extracted proteins were preliminarily quantified using a NanoDrop microvolume UV-Vis spectrophotometer set at 280 nm. A Bradford assay was carried out in order to determine protein concentration for the SazCA-INPN mineralisation module. A low working range (1-25µg/mL) microplate protocol was followed according to NanoDrop values. Working volumes of standard/sample was 150µL, with an equal volume of Bradford Protein Assay Reagent (ThermoFisher). Absorbance was measured at 595 using a microplate reader. Average protein concentration was determined to be 68.34 µg/mL (0.5 mM ZnSO4 + 0.5 mM IPTG).

Image 1
In Vitro Mineralization: To test the ability of engineered BL21(DE3) E. coli strain to precipitate CaCO3, we performed an in vitro mineralisation assay, adapting Zhu, et al. 's technique. Bacteria were cultured overnight in 30mL of LB +Kanamycin medium and 0.5 mM ZnSO4 at 25℃. IPTG induction was performed 3 hours prior to experimentation. The assay was run on 8 mL Tris-HCl buffer 8.3 and 50mL of saturated CO2 aqueous solution at 0℃. 3 mL of cell pellet were introduced into the solution, and the reaction was allowed to proceed on ice for an hour. At this point, the bacteria should have been able to produce bicarbonate ions. Cells were removed from the solution by centrifugation (15 min x 5000g). 25mL of a 0.3M solution of CaCl2 was added to the remaining supernatant as a calcium source. The reaction was left to run at 25℃ for 12h. Samples were filtered using vacuum filtration and dried at 50℃ to evaporate the solvent. Solid mass was weighed and recorded as “Wet Weight”. Upon preliminary analysis of FT-IR data, it was concluded that the mineral sample contained a large amount of water, elucidated by the stretching O-H peak at 3400 cm.1. Hence, the sample was dried further in liquid nitrogen for 48 hours, final weight was recorded at 2.3 g (yield=306.17%). Precipitated dry crystals were analysed using ATR-IR and 13C NMR. Enzymatic activity of SazCA: To measure the activity of the SazCA construct, a colorimetric Wilbur Anderson assay was adapted from Kim & Jo, (2022). The assay measures the ability of carbonic anhydrase to hydrate CO2. Protons released during the hydration reaction cause a decrease in the pH of the solution. Such displacement of H+ can be recorded as a function of time taken for pH to shift from 8.5 to 6.5. The colorimetric approach taken for the assay indirectly measured the change of pH by recording the colour change of phenol red upon the addition of SazCA. A reaction buffer of 20mM Tris pH. 8.3 (pKa=8.1) and 100µM phenol red (pKa=7.9) was used. Phenol red was chosen as the pH indicator as it shifts colours from yellow to pink over a pH range of 6.3 to 8.3. Total reaction volume was 1mL. 10µL of SazCA-BL21(DE3) culture were added into a cuvette for each reaction.  Varying amounts of saturated CO2 aqueous solution (0.279M) were added, volume filled to 1mL with corresponding amounts of reaction buffer. Data collection was performed by UV-Vis spectrophotometry, measuring absorbance change at 570 nm using the kinetics function of the spectrophotometer, recording every 0.1s for 30 minutes.  All reactions were performed ice-cold. The blank reaction consisted of 600µL reaction buffer and 400µL CO2 solution. SazCA samples compared to WT BL21. Abs values were obtained as a colorimetric reference for the reaction buffer adjusted for pH at 8.3, 7.5, and 6.5. Wilbur-Anderson Units (WAU) were calculated according to the following formula: WAU=(t0-t)/t, where t0 is the time (s) taken for the control to undergo a colour shift, and t is the time taken for SazCA samples to undergo a colour shift. Sequence and Features BBa_K4665005 SequenceAndFeatures ===References=== Anbu, P. et al. (March 1, 2016). Formations of calcium carbonate minerals by bacteria and its multiple applications. Springerplus 5(250). https://doi.org/10.1186/s40064-016-1869-2 Chaparro-Acuña, S.P., et al. (June, 2018). Soil bacteria that precipitate calcium carbonate: mechanism and applications of the process. Acta Agronómica 67(2). https://doi.org/10.15446/acag.v67n2.66109 De Luca, V. et al. (March 15, 2013). An α-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction. Bioorganic & Medicinal Chemistry Letters, 21(6): 1465.1469. https://doi.org/10.1016/j.bmc.2012.09.047 De Simone, G., et al. (May 1, 2015). Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense. Bioorganic & Medicinal Chemistry Letters, 1;25(9): 2002-2006. https://doi.org/10.1016/j.bmcl.2015.02.068 Dhami, N.K., et al. ( May 2013). Biomineralization of calcium carbonate polymorphs by the bacterial strains isolated from calcareous sites. Journal of Microbiology and Biotechnology, 23(5): 707-714. https://doi.org/10.4014/jmb.1212.11087 Hartmann, S., et.al. (January 22, 2022). Structure and protein-protein interactions of Ice Nucleation Proteins drive their activity. BioRxiv. https://doi.org/10.1101/2022.01.21.477219 Kim, J. H., & Jo, B. H. (2022). A Colorimetric CO2 Hydration Assay for Facile, Accurate, and Precise Determination of Carbonic Anhydrase Activity. Catalysts, 12(11), 1391. MDPI AG. http://dx.doi.org/10.3390/catal12111391 Pan, S. H., & Malcolm, B. A. (2000). Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). BioTechniques, 29(6), 1234–1238. https://doi.org/10.2144/00296st03 Singh, P., et al.. (2013). Effect of signal peptide on stability and folding of Escherichia coli thioredoxin. PloS one, 8(5), e63442. https://doi.org/10.1371/journal.pone.0063442 Zhu, Y., et.al (December 6, 2021). Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralisation. Synthetic and Systems biotechnology, 7(1): 460-473. https://doi.org/10.1016%2Fj.synbio.2021.11.008