Team:USP-EEL-Brazil/Experiments
Experimentos
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_0030) 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.
Our synthetic DNA were synthetized in gBlocks. For the gene 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 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
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.
Looking at the results, 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.
We cultivate the positive colonies above overnight for production of plasmids. The plasmid was purificated using Miniprep (Promega)
The agarose gel confirmed the presence and integrity of plasmids. These plasmids were quantitated using the NanoDrop OneC equipment and then 10 μL of the Miniprep product was lyophilized for submission of parts to the Registry.
Expression and Purification
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Biochemical Characterization
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