iGEM Patras 2021


There is something to be learned from project PGasus.

SDM (site-directed mutagenesis) is a procedure for designing specific, targeted modifications in plasmid DNA. This method can select or screen for mutations (at the DNA, RNA, or protein level) that have a desired, anticipated, or unknown effect through designing custom oligonucleotide primers to confer a specific mutation in a double-stranded DNA plasmid. The most extensively used methods use inverse PCR using standard primers to integrate mutations into the plasmid without requiring any modifications or unique strains.

We use a back-to-back primer design strategy, which enables exponential amplification to produce substantially more of the desired product. Furthermore, because the primers do not overlap, deletions are restricted only by the size of the plasmid, while insertions are limited only by the restrictions of conventional primer synthesis.

The novel enzymatic allelic variant constructs were synthesized using the plasmid carrying wild-type CYP2D6 and CYP2C19 cDNA as a template. Because all of the assessed variations are substitutions, the mutagenic sites were positioned as a miss-match on the forward primer.

To directionally clone a blunt-end PCR product into a vector, the pENTR Directional TOPO Cloning approach uses a highly efficient 5-minute cloning strategy ("TOPO Cloning"). All the pENTR/D-TOPO vectors cloned with the wild-type CYP2D6, CYP2C19, CPR and cytochrome b5 cDNAs were added to NEB 5-Alpha High-Efficiency E. coli cells.

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The pcDNA3.4-TOPO ΤΑ vector allows the rapid TOPO cloning of a PCR product containing a gene of interest downstream of the CMV promoter for high-level expression of the native protein in adherent mammalian cell cultures. All the gene variants were subcloned into a pcDNA3.4 vector with TOPO TA cloning sites.

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# Name Type Description Length
1 BBa_K3839000 Primer Forward mutagenic primer CYP2D6 L61S 21
2 BBa_K3839001 Primer Reverse primer CYP2D6 L61S 27
3 BBa_K3839002 Primer Forward mutagenic primer CYP2D6 F112S 21
4 BBa_K3839003 Primer Reverse primer F112S 19
5 BBa_K3839004 Primer Forward mutagenic primer S476I 21
6 BBa_K3839005 Primer Reverse primer CYP2D6 S476I 19
7 BBa_K3839006 Primer Forward mutagenic primer L15P 23
8 BBa_K3839007 Primer Reverse primer CYP2C19 L15P 24
9 BBa_K3839008 Primer Forward mutagenic primer CYP2C19 E274G 21
10 BBa_K3839009 Primer Reverse primer CYP2C19 E274G 18
11 BBa_K3839010 Primer Forward mutagenic primer CYP2C19 N286D 24
12 BBa_K3839011 Primer Reverse primer CYP2C19 N286D 20
13 BBa_K3839012 Primer Forward mutagenic primer CYP2C19 D293G 21
14 BBa_K3839013 Primer Reverse primer CYP2C19 D293G 27
15 BBa_K3839014 Primer Forward mutagenic primer CYP2C19 T304A 21
16 BBa_K3839015 Primer Reverse primer CYP2C19 T304A 18
17 BBa_K3839016 Primer Forward mutagenic primer CYP2C19 I327V 21
18 BBa_K3839017 Primer Reverse primer CYP2C19 I327V 19
19 BBa_K3839018 Primer Forward mutagenic primer CYP2C19 L380P 21
20 BBa_K3839019 Primer Reverse primer CYP2C19 L380P 21
21 BBa_K3839020 Primer Forward mutagenic primer CYP2C19 L413M 21
22 BBa_K3839021 Primer Reverse primer CYP2C19 L413M 21
23 BBa_K3839022 Primer Forward mutagenic primer CYP2C19 F487S 21
24 BBa_K3839023 Primer Reverse primer CYP2C19 F487S 21
25 BBa_K3839024 Coding CYP2D6-204 transcript 2367
26 BBa_K3839025 Coding CYP2C19-201 transcript 1473
27 BBa_K3839026 Coding CYB5A_202 transcript 405
28 BBa_K3839027 Coding POR-218 Transcript (CPR) 2043
29 BBa_K3839028 Primer CYP2D6 TOPO site Forward Primer 20
30 BBa_K3839029 Primer CYP2D6 TOPO site Reverse Primer 22
31 BBa_K3839030 Primer CYP2C19_201 TOPO site Forward Primer 26
32 BBa_K3839031 Primer CYP2C19_201 TOPO site Reverse Primer 30
33 BBa_K3839032 Primer POR-218 TOPO site Forward Primer 23
34 BBa_K3839033 Primer POR-218 TOPO site Reverse Primer 20
35 BBa_K3839034 Primer CYB5A_202 TOPO site Forward Primer 19
36 BBa_K3839035 Coding pENTR/D-TOPO 2580
37 BBa_K3839036 Primer CYB5A_202 TOPO site Reverse Primer 23
38 BBa_K3839038 Plasmid pENTR/D-TOPO vector + CYP2D6 4947
39 BBa_K3839039 Plasmid pENTR/D-TOPO vector + CYP2C19_201 4053
40 BBa_K3839040 Plasmid pENTR/D-TOPO vector + POR-218 4623
41 BBa_K3839041 Plasmid pENTR/D-TOPO vector + CYB5A_202 2985
42 BBa_K3839042 Plasmid pcDNA3.4-TOPO + CYP2D6_204 8403
43 BBa_K3839043 Plasmid pcDNA3.4-TOPO + CYP2C19_201 7509