Difference between revisions of "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 lligate 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 following: 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 ligate to the site of the Xbal of our gene, making he “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.

Clonig

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 One^C 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:

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 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

_{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, wihich 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

_{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.

The purification of LacTT will be performed using the AKTA PURE equipment and will be two steps: first, affinity chromatography 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 affinity for Nickel and will be retained in the Nickel column, as well as other proteins that may have affinity with Nickel. This way it is possible to separate most of the proteins present in the medium.

Affinity chromatography - Nickel columm

With the eluted fractions containing the LacTT the gel filtration was done

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.

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.

(colocar gráfico do bradford aqui)

OD 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 and the result was 0.109 mg / ml.

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.

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.

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.