Preparation of parts HygR-LHRZ and ZeoR-LHRZ

Dariusz Abramczyk

Jul 16, 2023

Preparation of parts HygR-LHRZ and ZeoR-LHRZ

DOI

https://dx.doi.org/10.17504/protocols.io.4r3l271o3g1y/v1

Dariusz Abramczyk¹

¹University of Edinburgh

Dariusz Abramczyk

University of Edinburgh

DOI: https://dx.doi.org/10.17504/protocols.io.4r3l271o3g1y/v1

Protocol Citation: Dariusz Abramczyk 2023. Preparation of parts HygR-LHRZ and ZeoR-LHRZ. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l271o3g1y/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: In development

We are still developing and optimizing this protocol. This is a protocol for preparation of a double-feature part (antibiotic selection marker- LHRZ)

Created: March 09, 2023

Last Modified: July 16, 2023

Protocol Integer ID: 78443

Keywords: lhrz this cloning protocol, preparation tandem pair hygr, insertion array before pichia transformation, combinatory assembly system for preparation, lhrz tandem pair, combinatory assembly system, homology recombination region, recombination process, cloning protocol, antibiotic selection marker, insertion array, pichia transformation, swapping hr, lhrz, hr event while the lhrz, preparation of parts hygr

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 HygR-LHRZ or the ZeoR-LHRZ 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

Protocol materials

ReagentBamHI-HF - 50,000 unitsNew England BiolabsCatalog #R3136L 
ReagentKpnI-HF - 20,000 unitsNew England BiolabsCatalog #R3142L 
ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S 
ReagentCarbenicillin 
ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g 
ReagentPrimeSTAR GXL PremixCatalog #R051B 
ReagentBamHI-HF - 10,000 unitsNew England BiolabsCatalog #R3136S 
ReagentSalI-HF - 2,000 unitsNew England BiolabsCatalog #R3138S 
ReagentGibson Assembly Cloning Kit - 10 rxnsNew England BiolabsCatalog #E5510S 
ReagentAsiSI - 2,500 unitsNew England BiolabsCatalog #R0630L 
Reagent1 g Carbenoxolone DisodiumbiorbytCatalog #orb320280 

Troubleshooting

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

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

PCR preparation part LHRZ for cloning with KpnI/BamHI (3'-end of LHRZ with
 BsaI or BsmBI RE site delivered by an oligo)
Duration02:00:00    

ReagentPrimeSTAR GXL PremixCatalog #R051B  
Aexpected size 998bpexpected size 998bp
components
LHRZ for (BsmBI)LHRZ for (BsaI)
eDA8 (125pg/uL)11
oligo 3101.251.25
oligo3171.25
oligo 3181.25
PrimeStar Premix 2x25
waterup 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
 
ABC
310 (F)LHRZ (BamHICCGGATCCCCGTGTAGGTCTACAAACTGG
318 (R)LHRZ  (KpnI-BsmBI-AsiSI)GCGGTACCCGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG
317 (R)LHRZ  (KpnI-BsaI-AsiSI)GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG
  
Programtimenumber of cycles
98oC10 sec33 x
55oC10sec
68oC1min 30 sec
4oChold
 

Duration00:30:00   
PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit
Elution with water 50uL

30m

KpnI and BamHI digestion of PCR product and pUC19 (all enzymes NEB)  BamHI/ KpnI
ReagentKpnI-HF - 20,000 unitsNew England BiolabsCatalog #R3142L  
ReagentBamHI-HF - 50,000 unitsNew England BiolabsCatalog #R3136L  

ABCD
10 x CutSmart888
BamHI-HF (20u/uL)1.51.51
KpnI-HF (20u/uL)1.51.51
PCR LHRZ 310/31750
PCR LHRZ 310/31850
pUC19        
 298 ng/uL10
waterup to 80 ->
RE digestion reactions
 Temperature37 °C   Duration04:00:00   

Duration00:30:00   
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

30m

Ligation reactions T4 DNA ligase (NEB)
 ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S  
A12
pUC B/K ~100ng/uL) BamHI/KpnI11
T4 DNA ligase11
LHRZ (100ng/ul)  B/K(310/317)5.5
10 X buffer11
waterup to 10 ->
LHRZ (100ng/ul)(310/318) B/K5.5
 DurationOvernight 16oC  

Duration02:00:00   

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL ReagentCarbenicillin  and growth at DurationOvernight   Temperature37 °C   

Duration01:00:00   
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
E.coli colony PCR 
Positively verified clones re-cultured in LB + 100 ug/mlReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g   for miniprep (plasmid preparation)DurationOvernight   

Plasmid isolation (miniprep) Qiagen miniprep Duration01:00:00   
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

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
 ReagentPrimeSTAR GXL PremixCatalog #R051B  
componentsZeoRHygR
eDA48 (175 (ng/uL)1
eDA78 (125pg/uL)1
oligo 3081.25
oligo3091.25
oligo 3061.25
oligo 3071.25
PrimeStar Premix 2x2525
waterup to 50up to 50uL
  
ABC
308 (F)Zeocin cassette (SalI-BsmBI) overhang GTAGCTGTCGACCGTCTCAgtagCCCACACACCATAGCTTCAAAATG
309(R)Zeocin cassette (BamHI)GGGATCCGCAAATTAAAGCCTTCGAGCG
306 (F)HygR (SalI-BsaI)CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCC
307 (R)HygR (BamHI)GCGGATCCCAGTATAGCGACCAGCATTCACATAC
 

Duration00:30:00   
PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit
Elution with water 50uL

30m

SalI and BamHI digestion of PCR product and pUC19 (all enzymes NEB)BamHI
ReagentBamHI-HF - 10,000 unitsNew England BiolabsCatalog #R3136S  
ReagentSalI-HF - 2,000 unitsNew England BiolabsCatalog #R3138S  
 
componentsBCDE
10x cutsmart buffer NEB8888
BamHI-HF (20u/uL)1.51.51.51.5
eDA99          70 ng/uL30
eDA101        81 ng/uL30
PCR hygR 306/30750
PCR zeo 308/30950
SalI-HF (20u/uL)1.51.51.51.5
waterup to 80 ->
          Temperature37 °C   Duration04:00:00   
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            

Duration00:30:00   
PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit
Elution with water 50uL
loaded 2 ul of each fraction

30m

Ligation reactions T4 DNA ligase (NEB)

 ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S  
T4 DNA ligase11
10 X buffer11
eDA101 (S/B)3
zeo 308/309 (S/B)5
eDA99 (S/B)3
hygR 306/307 (S/B)5
waterup to 10 ->
  DurationOvernight 16oC  

Duration02:00:00   

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB + 100ug/mL carbenicillin and growth at DurationOvernight   Temperature37 °C   
ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g  

Duration01:00:00   
.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
 
All colonies verified positively
Positively verified clones re-cultured in LB ampicillin for miniprep (plasmid preparation)DurationOvernight    

Plasmid isolation (miniprep) Qiagen miniprep Duration01:00:00   
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.
  

 Duration01:30:00   PCR amplification for further Gibson assembly reaction
 ReagentPrimeSTAR GXL PremixCatalog #R051B  
 PrimeStarHS 2x premix (Takara) High Fidelity
components
for Gibson 1for Gibson 2
waterup to 50 uLfor Gibson 1
Primestar2x2525
oligos 327/3201.25/1.25
oligos 319/3261.25/1.25
eDA103 (74pg/uL)1.51.5
 
PROGRAM TAK-2.30time reactioncycles
98C10sec33x
55C10 sec
68C2min 30 sec
4Chold
   PCR reaction treatment with DpnI for Duration00:30:00   at Temperature37 °C   

Duration00:30:00     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)

30m

Gibson assembly of 3.3kb fragment 1 and 1.9kb fragment 2
ReagentGibson Assembly Cloning Kit - 10 rxnsNew England BiolabsCatalog #E5510S  
 Duration00:30:00   Temperature37 °C   
AB
Gibson1 319/326 (3.3kb) 40nM 3.3 kb1
Gibson2 327/320 (2.05 kb) 40nM1
Gibson 2x mix2.5
water0.5
 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 DurationOvernight   Temperature37 °C   

30m

Single colonies verified by colony PCR
Duration01:00:00   
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  

Positively verified clones re-cultured in LB + 100 ug/m Reagent1 g Carbenoxolone DisodiumbiorbytCatalog #orb320280   for miniprep (plasmid preparation)DurationOvernight    

Plasmid isolation (miniprep) Qiagen miniprep Duration01:00:00   

Restriction digestion verification. Plasmid eDA115  eda115.dna   digested with AsiSI (NEB)
ReagentAsiSI - 2,500 unitsNew England BiolabsCatalog #R0630L  
 
ABC
AsiSI 11
eDA115      326 ng/uL3
eDA103      103 ng/uL8
10 x CutSmart33
waterup 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  

A	expected size 998bp	expected size 998bp
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

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


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

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 ->

A	1	2
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

components	ZeoR	HygR
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

components	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 ->



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 ->

components	for Gibson 1	for Gibson 2
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

PROGRAM TAK-2.30	time reaction	cycles
98C	10sec	33x
55C	10 sec
68C	2min 30 sec
4C	hold

	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

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 ->

Public workspacePreparation of parts HygR-LHRZ and ZeoR-LHRZ

Preparation of parts HygR-LHRZ and ZeoR-LHRZ