FastDigest Mph1103I

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	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].

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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Manufacturer protocol

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