Team:USP-EEL-Brazil/Experiments
Molecular Biology
The molecular biology stage is the initial part of our project in relation to LabWork. To obtain the enzyme of interest, laccase, the following steps must be taken:
- Amplification of gene (PCR); (seria legal se cada um desses tópicos fossem redirecionados para os respectivos protocolos)
- Gene cloning (3A Assemlby);
- Bacterial transformation;
- Expression and purification of enzyme;
Here we will address the steps of amplification, cloning and transformation. To carry out the experiments you need the genetic material (primers, promoters, RBSs, genes, terminators, vectors, etc)
The genes we are working on, LacPL and LacPH, are constituted of RBS (BBa_B0030) and CDS. The promoter and the terminator were used of BBa_R0010 (Kit plate 3 Well 4G). The cloning was made inserting the genes into the BBa_R0010.
Our DNA was synthetized in gBlock format by IDT.
Image 1: Synthetic DNA from IDT.
Source: Personal archive
Gibsson Assembly
Our synthetic DNA were synthetized in gBlocks. For the gene to become functional we did Gibson Assembly to ligate the gBlocks.
Image 2:Electrophoretic profile of Gibson Assembly of LacPL and LacPH.
Source: Personal archive
Amplification (PCR)
Confirmed the Gibson assembly we amplified the ligation products by PCR.
Image 3: Electrophoretic profile of' amplification of LacPL and LacPH using diferents volume of template.
Source: Personal archive
Preparing the plasmids
Made the amplification of genes, we prepared the vector with LacI promoter (BBa_R0010) and linearized plasmide backbone pSB1C3 (2016) for insertion of genes. The BBa_R0010 was transformaded in DH5alpha for production of more plasmids.
Image 4: Electrophoretic profile of plamids from BBa_R0010.
Source: Personal archive
Ligation (3A Assembly)
With the plasmids and the genes ready, we did 3A Assembly to insert our gene. First, we inserted the gene into linearized plasmid pSB1C3 from USP-EEL BRAZIL Team 2016, because we do not have the DpnI enzyme. For this reason, we tried insert also into BBa_J04450
Image 5: Scheme of 3A Assembly to insert our part into linearized plasmid backbone.
Source: Personal archive
Image 6: Scheme of 3A Assembly using BBa_J04450 as pSB1C3 backbone.
Source: Personal archive
Simultaneously, we did 3A Assembly for insertion of the gene into BBa_R0010. The 3A Assembly protocol was modified as follows: The part “A” was cleaved using SpeI and PstI and the gene was cleaved by Xbal and Pstl. By making the ligation, the site for part A’s Spel ligates to the site of the Xbal of our gene, making the “M” point. Then the Pstl’s site of the plasmid of our gene ligate and close the plasmid.
Image 7: Scheme of 3A Assembly to insert our parts into BBa_R0010.
Source: Personal archive
Transformation and confirmation of the ligation (Colony PCR)
After this ligation step, it was transformed in E. coli DH5alpha and we did the colony PCR.
Image 8: Electrophoretic profile of colony PCR of LacPH.
Source: Personal archive
Looking at results, it was not possible to obtain positive colonies of LacPH.
Image 9: Electrophoretic profile of colony PCR of LacPL.
Source: Personal archive
Image 10: Electrophoretic profile of colony PCR of LacPL and LacPH.
Source: Personal archive
Looking at the results of imagen 9 and 10, we can confirm that the genes are in the colony with the gene LacPL in BBa_R0010 (Lacl promoter) and LacPL in the linearized vector and also LacPH in BBa_J04450.
Cloning
We cultivated the positive colonies above overnight for production of plasmids. The plasmid was purified using Miniprep (Promega).
Image 11: Electrophoretic profile of plasmids.
Source: Personal archive
The agarose gel confirmed the presence and integrity of plasmids. These plasmids were quantified using the NanoDrop OneC equipment and then 10 μL of the Miniprep product was lyophilized for submission of parts to the Registry.
Image 12: Image of the plate with lyophilized DNA for submission.
Source: Personal archive
Expression and Purification
The purification step is known as the step, which we obtain the laccases for further testing, characterization and its applications. For reasons of lack of time and material, we weren’t able to produce the laccases LacPL and LacPH. However, we could work on LacTT, gifted by Evandro José Mulinari. All the designed parts has LacI as promotor. So the expression will happen in presence of lactose or IPTG, therefore the culture medium of bacteria is a lactose-containing autoinducing medium. For each liter of medium there is:
Table 1: The autoinducting medium composition
|
(NH4)2SO4 |
3,3g |
|
KH2PO4 |
6,8 g |
|
Na2HPO4 |
7,1 g |
|
glycerol |
5,0 g |
|
glucose |
0,5 g |
|
lactose |
2,0 g |
|
tryptone |
10,0 g |
|
Yeast extract |
5,0 g |
Pre-inoculum
After preparing and sterilizing the autoinducing medium, the pre-inoculum was prepared. For each liter of autoinducing medium, 10 ml of pre-inoculum were prepared using 10 ml of LB medium + 10 μl of kanamycin + 10 μl of chloramphenicol and inoculated with 20 μl of the glycerinated stock of the transformant. The pre-inoculum was cultured overnight at 37 ° C at 180 rpm.
Production of biomass and expression
The next day 10 ml of pre-inoculum was inoculated into 1L of an auto-inducing medium. The culture was incubated at 37 ° C at 130 rpm. The optical density was monitored up to the value of 0.6 at 600 nm, which corresponds to the log phase of cell growth. Upon reaching the log phase, CuSO4 was added to the auto-inducing medium at a final concentration of 0.1 mM. After adding the salt the culture was incubated at 18 ° C and 130 rpm overnight.
Image 1: Culture after 16h since the addition of CuSO4.
Source: Personal archive.
Cell lysis
After 16 h of culture, the medium was centrifuged at 4000 rpm for 2 h to separate the cells. Cells were resuspended in cell lysis buffer. For each liter of culture, a maximum of 50 ml of buffer was used.
Image 2: Centrifugation and resuspension of cells.
Source: Personal arquive.
With the cells resuspended in the lysis buffer, the medium was sonicated to promote sonication-mediated lysis. The protocol of the device itself was used for cell lysis, which is: 40% of power and cycles of 30 seconds, totaling 15 minutes of sonication, that is, 30 minutes of operation.
Image 3: Sonication and cell lysis.
Source: Personal arquive.
Purification
After lysis, the proteins were available in the liquid phase. The solution was then clarified by centrifugation at 11000 rpm for two 45 min cycles.
Image 4: Separation of solid and liquid phase.
Source: Personal arquive.
The two tubes on the left are the cells pellets and the two tubes on the right are the clarified solution containing the proteins solubilized in the liquid phase. The clarified solution is then concentrated to a volume of 10 mL to proceed to the purification step.
Image 5: Concentration of extract.
Source: Personal arquive.
The purification of LacTT will be performed using the chromatography system equipment AKTA PURE and will be two steps: first, affinity using Nickel column and the second step will be gel filtration. LacTT was designed in such a way that the protein contains HisTag. HisTag has an affinity for Nickel and will be retained in the Nickel column, as well as other proteins that may have an affinity with Nickel. This way it is possible to separate most of the proteins present in the medium.
Affinity chromatography - Nickel columm
Image 6: Nickel columm coupled in the equipment AKTA PURE chromatography system.
Source: Personal arquive.
Image 7: Eluate fraction collector.
Source: Personal arquive.
Image 8: Affinity chromatography profile of LacTT.
Source: Personal arquive.
Looking at chromatography profile we took the fraction corresponding to the peaks: fractions 4, 5 and 7 for the biggest peak; fractions 24, 25, 31, 33 for the other peaks and fraction 46 to check if there are no proteins present, using the protein electrophoresis technique.
Image 9: Protein electrophoresis profile of LacTT.
Source: Personal arquive.
The molecular weight of the LacTT with HisTag is around 80 kDa. Analyzing the electrophoresis and chromatography profiles we took the elution fraction from number 30 to 38 to carry out the gel filtration.
Gel filtration chromatography
With the eluted fractions containing the LacTT the gel filtration was done
Image 10: Superdex 200 columm coupled in the equipment AKTA PURE chromatography system.
Source: Personal arquive.
Image 11: Gel filtration chromatography profile of LacTT.
Source: Personal arquive.
Looking at chromatography profile we took the fraction from 17 to 36 that corresponds to the peak. Protein electrophoresis technique was used to verify the presence of the LacTT.
Image 12: Protein electrophoresis profile of gel 1.
Source: Personal arquive.
Image 13: Protein electrophoresis profile of gel 2.
Source: Personal arquive.
We collected the fractions containing the laccase and concentrated in a final volume of 10 ml. And so we obtained purified laccase to proceed with biochemical characterization.
Image 14: Image of the purified LacTT.
Source: Personal arquive.
LacPL and LacPH do not have HisTag. Therefore, purification will be done first by ion exchange chromatography. Knowing the isoelectric point of the enzymes is possible to carry out the purification. After the ion exchange chromatography will be done gel filtration.
Biochemical Characterization
At this biochemical characterization step, we’ll do enzymatic tests to find the optimal pH and temperature of the enzyme, as well as the activity (using ABTS substrate) and inhibitory activity of some compounds like F- and Cl-. Furthermore, it will be tested interferers degradation of endocrine disruptors. Due to lack of time and materials, it has been done the LacTT characterization. For the LacPL and LacPH characterization, it will be used the same tests.
Quantification of protein
Firstly, it’s needed to quantify the enzyme. Two measurements were taken: bradford method and using nano drop.
Image 1: Bradford cure. Source: Josman Velasco.
The OD of our sample was 0.7664. Isolating the X from the straight equation, we have 112 μg / mL protein.
The measured protein concentration by NanoDrop OneC at wavelength 280 nm was 109 μg / ml.
Image 2: Determination of protein concentration by equipment NanoDrop OneC.
Source: Personal arquive.
Analyning both of results of concentration is possible to conclude that the values are very close, with percentual error of 2,7%
Caracterization
For the tests, it was assembled the reaction:
- 160 uL of phosphate-citrate-glycine buffer 100 mM (pH depends on the test)
- 20 uL of enzyme;
- 20 uL of ABTS 1mM;
Laccase oxides the ABTS forming a blue compound. Its absorbance was measured at 420 nm on spectrophotometer.
pH assay
Thielavia terrestris is a thermophilic fungus, so the optimum pH determination assays were conducted at 60 ° C. It was tested pH from 2.0; 2.5; 3.0; 3.5; 4.0; 4,5; 5.0; 5.5; 6.0; 6.5; 7.0 and 7.5 utilizando 100 mM citrate phosphate glycine buffer.
Image 3: The effect of pH on the relative activity of LacTT.
Source: Personal arquive.
The optimum pH for LacTT activity is pH 4,0. For the effluent treatment if interesting a pH around 7,0. However, in the pH 7,0, the LacTT do not have a good activity.
Temperature assay
It has been determined that the optimal pH for the laccase of Thielavia terrestris is 4.0. Knowing the optimum pH proceeded for the optimum temperature test. Using pH 4.0 tests were performed at temperatures of 30; 35; 40; 45; 50; 55; 60; 65; 70; 75 and 80 °C.
Image 4: The effect of temperature on the relative activity of LacTT.
Source: Personal arquive.
The optimum temperature for LacTT is 55°C. Knowing the pH and optimum temperature, tests were carried out to determine the maximum activity using ABTS.
Image 5: µmol of ABTS+ formed by reaction time.Source: Personal arquive.
With the equation of the linear region of the curve, the maximum laccase activity can be calculated.
Image 6: Linear region of the curve for determination of laccase activity. Source: Personal arquive.
Analyzing the linear equation (1) is possible obtaining the value of enzyme activity of 0,5 mU (µmol/min).
Tests were also carried out to verify the inhibition of laccase. According to Auriol (2007) fluoride, ion and chloride ion has inhibition activity in laccases. The reaction was assembled as follows:
- 150 µl of citrate fosfate glycine buffer pH 4,0;
- 20 µl of enzyme;
- 20 µl of ABTS;
10 µl of inhibitor;
Image 7: Test of inhibition using NaF. Source: Personal arquive.
Its possible to observe a little reduction in the activity has occurred, which confirms the partial inhibition of laccase by fluoride To the inhibition test with Cl- it was used ClO-. However, it wasn’t possible to conclude anything with this experiment, because the ClO- degrades the ABTS, discoloring the medium.
Image 8: Desmonstration of discoloring effect of hypochlolite on ABTS. a) ABTS without laccase and hypochlorite; b) oxidated ABTS after adding laccase; c) discoloration ov solution after added hypochlorite.
Source: Personal arquive.
We started the endocrine disrupters degradation tests. We did tests with Bisphenol A (BPA), Nonylphenol and ß-estradiol.
First, we looked on the literature to know about the maximum absorption wave length of each compound. The wave length for ß-estradiol is 280 nm (VERBINNEN; NUNES, 2010). For the nonylphenol, it is 277nm (AHEL; GIGER, 1985) and for BPA the wave length is 280nm.
With the wave length information, we carried ou tests of degradation of BPA and ß-estradiol according to the following reagents volumes:
- 160 µl of citrate fosfate glycine buffer pH 4,0;
- 20 µl of enzyme;
- 20 ul of substrate;
The reaction was assembled at 55 °C for 1 hour in a plate with 96 well to monitoring the absorbance.
Image 9: Test of degradation of BPA and ß-estradiol. Source: Personal arquive.
Analyzing the results were raised some hypotheses. LacTT degraded BPA resulting in decreased absorbance or LacTT had no activity on BPA and ß-estradiol.
To investigate whether the laccase has no activity at all, TLC and HPLC analyzes were performed.
Image 10: HPLC profile: a) Chromatography profile of buffer; b) Chromatography profile of BPA dissolved in buffer. Source: Personal arquive.
Unfortunately, it was not possible to detect BPA by HPLC. The peak shown in the chromatogram corresponds to the buffer. For TLC, it was possible to detect BPA, but the reaction chromatography profile was the same as the standard, indicating that it had neither degradation nor polymerization of the substrate.
Image 10: Thin layer chromatography. a) Chromatographic profile of different concentration of BPA. b) Chromatographic profile of degradation test. The arrow is indicating the bands corresponding BPA. There are no other bands. The dark spots on the bottom are laccase. Source: Personal archive.
Unfortunately no further experiments could be carried out, because it was not possible to express and purify the LacPL and LacPH due to the lack of time and resources. However, we intend to continue the characterizations after the Jamboree.
REFERENCES
AURIOL, Muriel et al. Laccase-catalyzed conversion of natural and synthetic hormones from a municipal wastewater. Water Research, [s.l.], v. 41, n. 15, p.3281-3288, ago. 2007. Elsevier BV. http://dx.doi.org/10.1016/j.watres.2007.05.008.
AHEL, M.;GIGER, W.; Determination of nonionic surfactants of the alkylphenolpolyethoxylate Type by High-Performance Liquid Chromatography, Analytical Chemistry, v. 57, p.2584-2590, 1985.
VERBINNEN, R. T.; NUNES, G. S.; VIEIRA, E. M. Determinação de hormônios estrógenos em água potável usando CLAE-DAD. Quimica Nova. v. 33, n.9, p. 1837-1842, 2010.