Biomod/2011/Slovenia/BioNanoWizards/methplasmidconst

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Plasmid construction


Overview

All of the ORFs constructed within the project were assembled into Novagen(R) pET41a(+) vector already comprising glutathione S-transferase (GST) sequence downstream of the T7 promoter, RBS and ATG (5929 bp). Genes leading to desired protein chimeras were amplified via PCR, isolated from agarose gel, cut at their respective restriction sites and ultimately cloned into MCS region of the plasmid after cutting it with NdeI and XhoI restriction enzymes. In case of GST-ZFP chimeras, vector was opened differently, using SpeI and XhoI restriction sites wherein ZFP DNA fragment was ligated. Protein expression was driven by IPTG inducible T7 promoter. 8x His was cloned in frame at the 3' end followed by stop codon and T7 terminator to facilitate protein purification. Having His tag at the 3' end of the open reading frame is especially useful since one can be sure that the whole protein was expressed during protein production if a band at correct position is observed on a Western blotting membrane.


Gene sources

Genes encoding ZFPs were synthesized by GeneArt, plasmid encoding MBP was obtained from Addgene and GST was already part of the vector. mCitrine and Renilla luciferase genes were available in house. Primers for PCRs were ordered from Sigma.


Linker selection

GST-ZFP and MBP-ZFP chimeras were designed to have a 3 amino acid long linker [GSG] in between ZFP and the solubility tag. Twin ZFP chimeras were similarly designed, meaning again linked to the MBP solubility tag with [GSG] amino acid linker on both N- and C- terminal MBP ends. After cloning using restriction enzymes with 6 bp recognition sequence additional 2 amino acids were introduced at each linker site. In contrast, 10 AA linker was used for assembling RLuc-2C7 and YFP-AZPA4 chimeras with the following AA sequence [GGSGGGSGGS] again adding additional 2 amino acid residues after successful cloning. MBP was cloned to the 5' end of RLuc-2C7 and YFP-AZPA4 leaving behind four amino acids long linker in the form of [GSGS].


Cloning procedure

(1) PCR reaction with a set of two sequence specific primers, one forward and one reverse (ordered via Sigma)
(2) PCR verification on an agarose gel. Excision of bands from the gel and purification of the PCR product.
(3) Vector and PCR product restriction using specific restriction enzymes.
(4) Single gene ligation or 3-point ligation of two genes into the pre-cut vector using T4 DNA ligase.
(5) Addition of ligation mixture to E. coli DH5α cells. Incubation for 30 min.
(6) Heat shock for ~3-4 min.
(7) After adding 1 ml of LB medium to the cells they are shaked for ~ 1 h and thereby recovered from heat shock.
(8) Cells are then collected by spinning the tubes for 3 min at 7000 rpm using tabletop microcentrifuge and finnally spread over LB kanamycin agar plate.

Schematically, cloning was conducted as depicted on the scheme below:


Figure 48: Cloning overview of the ZFP fusions constructed during the project. See text above for details. Length of the rectangles depicting genes used for cloning roughly corresponds to their actual size.
Here is a link to a document containing all the produced proteins' nucleotide and amino acid sequences.
 

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