Team:Saint Joseph/Experiments
Experiments
Strategy
Provide the laccase enzyme to be directed to periplasmic area and increase the total amount of purified enzyme from E. coli.
Laccase is an enzyme which cleans textile waste water by decolorization. We used two methods: We designed the laccase enzyme produced by a white-rot fungi called Trametes versicolor bioinformatically.
Why Trametes versicolor?
Trametes versicolor is a white-rot fungi. Laccase has multiple different sources but studies show that laccase produced by Trametes versicolor has wide-range usage area. The activity of enzyme is higher than the laccases produced from other organisms
Why DSB region?
PelB sequence is added to investigate if the activity of laccase increases when the enzyme is secreted to the periplasmic region.
Why PelB signal sequence?
PelB sequence is added to investigate if the activity of laccase increases when the enzyme is secreted to the periplasmic region.
Why E.coli SHuffle Strain?
E.coli SHuffle strain promotes disulfide bond formation in cytoplasm. Disulfide bonds increase the activity of the enzymes.
Methodology
Bioinformatic Design
Plasmids used in the studies are designed with Benchling programme
Recombinant DNA Construction
Cloning vector and laccase gene are transformed into E.coli DH5α strains and produced separately. After production they are isolated and cut with restriction enzymes. Cloning vector and laccase gene are ligated and finally laccase gene is cloned.
Protein Expression
Cloned laccase genes are isolated and then transformed into expression vector to obtain protein. After expression step, proteins are isolated.
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1. WILD TYPE laccase
We optimised it because we want to avoid codon degeneration. We cloned this enzyme in E.coli DH5α and we produced it in E.coli SHuffle strain. Once the enzyme got produced in SHuffle, we isolated the enzyme and tested it.
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2. pelB + LACCASE
We added pelB signal sequence to laccase gene, and we requested our designed sequence in a cloning plasmid from IDT.
We transformed this plasmid to E. coli DH5α for cloning the sequence. We transferred cloned sequences to SHuffle strain for expression of the laccase enzyme. The enzyme is secreted from the cytoplasm to the periplasm.
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3. DSB + LACCASE
We added the disulphide bonding isomerase (DSB) sequence to laccase gene, and we requested our designed sequence in a cloning plasmid from IDT.
We transformed this plasmid to E. coli DH5α for cloning the sequence. We transferred cloned sequences to SHuffle strain for expression of the laccase enzyme. DSB region is added to wild-type laccase for increasing disulphide bond formation and enzyme activity.
Our Plasmid
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E.Coli SHuffle
The laccase enzyme has disulfide bonds in its wild type form. Laccase with these disulfide bonds cannot be produced in E.Coli DH5α. That is why we transformed it to the SHuffle strain to produce the laccase enzyme. This strain can form disulfide bonds of the protein naturally so we do not need to induce disulfide bond formation. E.Coli DH5α is only used for cloning.
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Isolation of Laccase
The designed laccase enzymes has histidine tags at the end. The isolation kit we got contains nickel particles. These particles recognize and bind to histidine tags. We placed this mixture on a magnetic tube stand. Enzymes stick to the wall with the magnet and the remaining liquid, the supernatant, is discarded. The remained pellet is our enzyme.
After isolating the enzyme produced in SHuffle , we stored it at -20 oC. Then we interacted these enzymes with dyed water.
Measurement Kit
We did the measurement experiment with iGEM Kit.
Measurement Kit
Plasmid Isolation Protocol with Kit
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1- Collection of bacteria: E.coli in 1.5 ml micro-centrifuge pellet is taken to overnight culture in 1.5-5 ml LB medium containing appropriate antibiotic.
Note: Solution 1,2,3 allows cell membrane disintegration because it contains SDS, it breaks down the lipids in the cell membrane. It removes the proteins.
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2- Resuspension: We added 100 micro-liters of “solution 1 ”and we resuspended the bacterial palette with vortex.
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3-Lysis: We added 200 micro-liters of “solution 2”, and mixed slowly by inverting the micro-tubes 4-5 times, keeping it at room temperature for 1 minute. If “VisualLyse” is added, the solution turns blue, stirring slowly until a homogeneous blue solution is obtained.
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4-Neutralization: We added 350 micro-liters of solution3. We slowly mix the micro-tube 4-6 times and let stand at room temperature for 1 minute. If Ly Visual Lyse ”is added, mix slowly until all blue marks are removed.
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5- Pellet Cell residues are centrifuged for 5 minutes at 10000 rpm in micro-centrifuge.
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6. DNA binding is transferred to the supernatant spin column. It is then centrifuged for 2 minutes and the flowing liquid discarded. Special bit tube with supernatant spin column filter.
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7- Washing: 750 micro-liters of Solution Wash Solution ”is added and centrifuged for 1 minute and then the flowing liquid is discarded.
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8- Wash the Column again.
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9. Centrifuge for 1 minute to remove any “Wash Solution” residue.
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10- Elution: Transfer the column to a new 1.5 ml micro-tube. Add 50 micro-liters of Elution Buffer to the center of the Spin Column and centrifuge at 10,000 rpm for 2 minutes. The elution buffer washes the plasmid DNA in the filter.