Difference between revisions of "Team:USP-EEL-Brazil/Experiments"
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• Expression and purification of enzyme;<br/> | • Expression and purification of enzyme;<br/> | ||
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| − | <p style=" margin-bottom: 50px; text-align: justify; text-indent: 5%; ">Here we will address the steps of amplification, cloning and transformation. To carry out the experiments | + | <p style=" margin-bottom: 50px; text-align: justify; text-indent: 5%; ">Here we will address the steps of amplification, cloning and transformation. To carry out the experiments we need the genetic material (primers, promoters, RBSs, genes, terminators, vectors, etc)<br/> |
</p> | </p> | ||
<p style=" margin-bottom: 50px; text-align: justify; text-indent: 5%; ">Our part are constituted of RBS and gene. The promoter and the terminator were used of BBa_R0010 (Kit plate 3 Well 4G). The cloning was made inserting our part into the BBa_R0010. Our DNA was synthetized in gBlock format by IDT. <br/> | <p style=" margin-bottom: 50px; text-align: justify; text-indent: 5%; ">Our part are constituted of RBS and gene. The promoter and the terminator were used of BBa_R0010 (Kit plate 3 Well 4G). The cloning was made inserting our part into the BBa_R0010. Our DNA was synthetized in gBlock format by IDT. <br/> | ||
Revision as of 18:21, 11 October 2018
Experimentos
Molecular Biology
Some news this fine day!
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);
• 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 we need the genetic material (primers, promoters, RBSs, genes, terminators, vectors, etc)
Our part are constituted of RBS and gene. The promoter and the terminator were used of BBa_R0010 (Kit plate 3 Well 4G). The cloning was made inserting our part into the BBa_R0010. Our DNA was synthetized in gBlock format by IDT.
Our synthetic DNA were synthetized in gBlocks. For our part to become functional we did Gibson Assembly to lligate the gBlocks.
Confirmed the Gibson assembly we amplified the Gibson products by PCR.
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.
With the plasmids and the our part 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.
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.
After this ligation step, it was transformed in E. coli DH5alpha and we did the colony PCR.
Expression and Purification
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Biochemical Characterization
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