FastDigest Mph1103I

Restriction Enzymes NsiI / Mph1103I

Experiment
Restriction Enzymes NsiI / Mph1103I
Product
FastDigest Mph1103I from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Protocol tips
The resulting plasmid was cut with enzymes Mph1130I and NheI in order to ligate the gene into a Mph1130I‐ and NheI‐digested pNivC vector (with ampicillin resistance marker), where it was attached to ribosome binding site (RBS) ‘C' (AAGTTAAGAGGCAAGA) which allows a moderate translation rate [31].

Publication protocol

"Cloning and plasmid propagation were carried out in
E. coli DH5a. An E. coli codon adapted version of the
phosphoketolase gene from Bifidobacterium adolescentis
(pkt, UniProt: A1A185, previously used in multiple metabolic engineering studies [18,19,29]) was synthesized by Life
Technologies (Thermo Fisher Cambridge, MA, USA).
Optimization of codon usage was done using JCat [30],
while excluding recognition sites of restriction enzymes
involved in the cloning process: EcoRI, NheI, Mph1103I,
PstI, SalI, BcuI, XhoI. The codon optimized sequence is
shown in the Supporting Information. A HIS-tag was
added after the start codon. The PKT gene was amplified
by PCR using primers pkt-F and pkt-R and cloned into
cloning vector pJet (CloneJet PCR Cloning Kit, Thermo
Scientific). The resulting plasmid was cut with enzymes
Mph1130I and NheI in order to ligate the gene into a
Mph1130I- and NheI-digested pNivC vector (with ampicillin resistance marker), where it was attached to ribosome
binding site (RBS) ‘C’ (AAGTTAAGAGGCAAGA) which
allows a moderate translation rate [31]. The RBSC-PKT
segment was cloned into vector pZ-ASS using EcoRI and
PstI. pZ-ASS replicates under p15A, a medium-copy number origin of replication, while gene expression is controlled by the constitutive ‘strong promoter’ pgi-20 [32].
This ‘default’ combination of regulatory elements is a normal practice in our lab [33]; furthermore, a promoter with
lower expression strength (pgi-10) was found to hinder
PKT-dependent growth, indicating insufficient activity of
the heterologously expressed enzyme. A streptomycin resistance cassette allowed for selection. All restriction enzymes
used were FastDigest (Thermo Scientific)."

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Papers

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for FastDigest Mph1103I below.

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