Team:Patras/Parts

iGEM Patras 2021

Parts

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.

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.

#	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