Preparation of parts HygR-LHRZ and ZeoR-LHRZ

Dariusz Abramczyk

Published: 2023-07-17 DOI: 10.17504/protocols.io.4r3l271o3g1y/v1

Abstract

This cloning protocol is for preparation tandem pair HygR-LHRZ and ZeoR-LHRZ as a part of combinatory assembly system for preparation of integration/insertion arrays.

https://www.protocols.io/edit/nanochromosome-arrays-combinatory-assembly-cprkvm4w

Either the Hyg^R-LHR^Z or the Zeo^R-LHR^Z tandem pairs are designed to be use for all arrays and are displayed on 3'-end each of arrays.

During inchworming/swapping HR double cross-over recombination process, the antibiotic selection marker is replaced in every HR event while the LHRZ works as a homology recombination region used during all inchworming/swapping rounds.

(see https://www.protocols.io/edit/nanochromosome-arrays-combinatory-assembly-cprkvm4w)

Both parts are AsiSI free (this is for an optional excise site the cassette from the plasmid to isolate an insertion array before Pichia transformation)

General scheme for preparation library of AntR-LHRZ

Steps

Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3'-part

0.1.

Preparation of LHRZ - the first part of sequential cloning into pUC19

0.2.

PCR preparation part LHRZ for cloning with KpnI/BamHI (3'-end of LHRZ with

BsaI or BsmBI RE site delivered by an oligo)

2h 0m 0s

A	B	C
components	LHRZ for (BsmBI)	LHRZ for (BsaI)
eDA8 (125pg/uL)	1	1
oligo 310	1.25	1.25
oligo317		1.25
oligo 318	1.25
PrimeStar Premix 2x	25
water	up to 50

LHR(non-coding DNA) delivered from the DNA library design by dr Valentin Zulkover and synthesised by prof Adele Marston Adele Marston lab

A	B	C
310 (F)	LHRZ (BamHI	CCGGATCCCCGTGTAGGTCTACAAACTGG
318 (R)	LHRZ (KpnI-BsmBI-AsiSI)	GCGGTACCCGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG
317 (R)	LHRZ (KpnI-BsaI-AsiSI)	GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG

A	B	C
Program	time	number of cycles
98oC	10 sec	33 x
55oC	10sec
68oC	1min 30 sec
4oC	hold

0.3.

0h 30m 0s

PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit

Elution with water 50uL

0.4.

KpnI and BamHI digestion of PCR product and pUC19 (all enzymes NEB) BamHI/ KpnI

A	B	C	D
10 x CutSmart	8	8	8
BamHI-HF (20u/uL)	1.5	1.5	1
KpnI-HF (20u/uL)	1.5	1.5	1
PCR LHRZ 310/317	50
PCR LHRZ 310/318		50
pUC19 298 ng/uL			10
water	up to 80 ->

RE digestion reactions 37°C 4h 0m 0s

0.5.

0h 30m 0s

PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit

Elution with water 50uL

Checking DNA concentration

LHRZ 310/317 (BamHI/KpnI) - ~100ng/ul

LHRZ 310/318 (BamHI/KpnI) - ~100ng/ul

0.6.

Ligation reactions T4 DNA ligase (NEB)

A	B	C
pUC B/K ~100ng/uL) BamHI/KpnI	1	1
T4 DNA ligase	1	1
LHRZ (100ng/ul) B/K(310/317)	5.5
10 X buffer	1	1
water	up to 10 ->
LHRZ (100ng/ul)(310/318) B/K		5.5

0.7.

2h 0m 0s

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells

Selection on LB +100ug/mL and growth at 37°C

0.8.

1h 0m 0s

Several colonies obtained on both agar plates. Single colonies transferred on new LB +100ug/mL ampicillin plate, growth overnight and a single colonies submitted for bacterial colony PCR.

see protocol colony PCR

Material loaded on agarose gel for verification

Positively verified clones re-cultured in LB + 100 ug/ml for miniprep (plasmid preparation)

0.9.

Plasmid isolation (miniprep) Qiagen miniprep 1h 0m 0s

PCR verified and Sanger sequencing intermediate plasmid LHRZ(BsmBI)-pUC19 called eDA101eda101.dna

PCR verified and Sanger sequencing intermediate plasmid LHRZ(BsaI)-pUC19 called eDA99eDA99.dna

Preparation of ZeoR-LHRZ and HygR-LHRZ

The final step - insertion of ZeoR or HygR into plasmid eDA101 and eDA99 correspondingly

1.1.

PCR amplification of Zeocin expression cassette (BsaI-free) for cloning with BsmBI

Zeocin and hygromycin cassettes originally derived from plasmids pGS-GnTI and pGS-GnTII (Glycoswitch) glycoswitch plasmids

A	B	C
eDA48 (175 (ng/uL)		1
eDA78 (125pg/uL)	1
oligo 308	1.25
oligo309	1.25
oligo 306		1.25
oligo 307		1.25
PrimeStar Premix 2x	25	25
water	up to 50	up to 50uL

A	B	C
308 (F)	Zeocin cassette (SalI-BsmBI) overhang GTAG	CTGTCGACCGTCTCAgtagCCCACACACCATAGCTTCAAAATG
309(R)	Zeocin cassette (BamHI)	GGGATCCGCAAATTAAAGCCTTCGAGCG
306 (F)	HygR (SalI-BsaI)	CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCC
307 (R)	HygR (BamHI)	GCGGATCCCAGTATAGCGACCAGCATTCACATAC

1.2.

0h 30m 0s

PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit

Elution with water 50uL

1.3.

SalI and BamHI digestion of PCR product and pUC19 (all enzymes NEB)BamHI

A	B	C	D	E
10x cutsmart buffer NEB	8	8	8	8
BamHI-HF (20u/uL)	1.5	1.5	1.5	1.5
eDA99 70 ng/uL			30
eDA101 81 ng/uL				30
PCR hygR 306/307		50
PCR zeo 308/309	50
SalI-HF (20u/uL)	1.5	1.5	1.5	1.5
water	up to 80 ->

      `37°C` `4h 0m 0s`

1.4.

0h 30m 0s

PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit

Elution with water 50uL

1.5.

Ligation reactions T4 DNA ligase (NEB)

A	B	C

T4 DNA ligase	1	1
10 X buffer	1	1
eDA101 (S/B)		3
zeo 308/309 (S/B)		5
eDA99 (S/B)	3
hygR 306/307 (S/B)	5
water	up to 10 ->

1.6.

2h 0m 0s

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells

Selection on LB + 100ug/mL carbenicillin and growth at 37°C

1.7.

1h 0m 0s

.Several colonies obtained on both agar plates. Single colonies transferred on new LB +100ug/mL ampicillin plate, growth overnight and a single colonies submitted for bacterial colony PCR

see protocol colony PCR

(oligo 131/40 for verification ZeoR-LHRC-puC see plasmid map eda105.dna

(oligo 306/42 for verification HygR-LHRC-puC see plasmid map eda103.dna

Material loaded on agarose gel for verification

Positively verified clones re-cultured in LB ampicillin for miniprep (plasmid preparation)

1.8.

Plasmid isolation (miniprep) Qiagen miniprep 1h 0m 0s

PCR verified and Sanger sequencing intermediate plasmid ZeoR-LHRC-puC see plasmid eda105.dna

Tandem paired ZeoR-LHRZ part (BsmBI-free, AsiSI-free,) ready for cloning with BsmBI as a PCR template for a use in the protocol

https://www.protocols.io/edit/nanochromosome-arrays-combinatory-assembly-cprkvm4w

PCR verified and Sanger sequencing intermediate plasmid HygR-LHRC-puC see plasmid map eda103.dna The part is BsaI-free but contains internal AsiSI in hph gene , requires mutagenesis

Mutagenesis of AsiSI in eDA103 plasmid by Gibson assembly

Schematics of mutagenesis of AsiSI site in HygR by Gibson assembly.

Note

eDA103 contains two AsiSI sites, but only AsiSI site in HygR needs to be removed as a part of integration/insertion array. Another AsiSi site (in pUC19 vector) is not taking under a consideration.

2.1.

1h 30m 0s PCR amplification for further Gibson assembly reaction

PrimeStarHS 2x premix (Takara) High Fidelity

A	B	C
water	up to 50 uL	for Gibson 1
Primestar2x	25	25
oligos 327/320		1.25/1.25
oligos 319/326	1.25/1.25
eDA103 (74pg/uL)	1.5	1.5

A	B	C
98C	10sec	33x
55C	10 sec
68C	2min 30 sec
4C	hold

PCR reaction treatment with DpnI for 0h 30m 0s at 37°C

2.2.

0h 30m 0s PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit

Elution with water 50uL.

DNA Molar concentration of purified PCR products was calculated using bioline conventor DNA concentration convertor

Gibson 1 fragment 86 ng/uL

Gibson 2 fragment 46 ng/ul

PCR products verified on 1% agarose gel (4 uL loaded)

2.3.

Gibson assembly of 3.3kb fragment 1 and 1.9kb fragment 2

0h 30m 0s 37°C

A	B
Gibson1 319/326 (3.3kb) 40nM 3.3 kb	1
Gibson2 327/320 (2.05 kb) 40nM	1
Gibson 2x mix	2.5
water	0.5

Gibson assembly protocol protocol NEB Gibson Assembly Master Mix

Immediately after Gibson reaction is E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells

Selection on LB +100ug/mL ampicillin and growth at 37°C

2.4.

Single colonies verified by colony PCR

1h 0m 0s

Several colonies obtained on both agar plates. Single colonies transferred on new LB +100ug/mL ampicillin plate, growth overnight and a single colonies submitted for bacterial colony PCR

see protocol colony PCR

Oligos 41 and 307 used for primary verification.

Oligo sequences available in the plasmid eDA115 map eda115.dna

2.5.

Positively verified clones re-cultured in LB + 100 ug/m for miniprep (plasmid preparation)

2.6.

Plasmid isolation (miniprep) Qiagen miniprep 1h 0m 0s

2.7.

Restriction digestion verification. Plasmid eDA115 eda115.dna digested with AsiSI (NEB)

A	B	C
AsiSI	1	1
eDA115 326 ng/uL	3
eDA103 103 ng/uL		8
10 x CutSmart	3	3
water	up to 30 ->

Tandem paired Hyg-LHRZ part (BsaI-free, AsiSI-free,) ready for cloning with BsaI as a PCR template for a use in the protocol

https://www.protocols.io/edit/nanochromosome-arrays-combinatory-assembly-cprkvm4w

PreparationZeoR-LHRZ - cloning steps

29.

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing

Preparation of parts HygR-LHRZ and ZeoR-LHRZ

Abstract

Steps

Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3'-part

PreparationZeoR-LHRZ - cloning steps

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