TITLE: Preparation of parts HygR-LHRZ and ZeoR-LHRZ
AUTHORS: Dariusz Abramczyk

[DESCRIPTION]
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)

   



[STEPS]
SECTION: PreparationZeoR-LHRZ - cloning steps
29.  
   

SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.1. Preparation of LHRZ - the first part of sequential cloning into pUC19 
  
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.3. 30 min 
PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit
Elution with water 50uL
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.4. 
KpnI and BamHI digestion of PCR product and pUC19 (all enzymes NEB)  BamHI/ KpnI
  
  

 10 x CutSmart888BamHI-HF (20u/uL)1.51.51KpnI-HF (20u/uL)1.51.51PCR homC 310/31750PCR homC 310/31850pUC19 298 ng/uL10waterup to 80 ->
 37 °C 240 min 
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.5. 30 min 
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
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.6. Ligation reactions T4 DNA ligase (NEB)
   
   pUC-C 310/317pUC-C 310/31810 X buffer11HomC (310/317) B/K5.5HomC (310/318) B/K5.5pUC B/K11T4 DNA ligase11waterup to 10 ->
   


SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.7. 120 min 

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL   and growth at   37 °C 
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.8. 
60 min 
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)  
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
0.9. Plasmid isolation (miniprep) Qiagen miniprep 60 min 
PCR verified and Sanger sequencing intermediate plasmid LHRZ(BsmBI)-pUC19 called eDA101  
PCR verified and Sanger sequencing intermediate plasmid LHRZ(BsaI)-pUC19 called eDA99  

SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1. Preparation of ZeoR-LHRZ and HygR-LHRZ
  
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
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
   
components HLRZ (BsmBI)HLRZ (BsaI)eDA8 (125pg/uL)11oligo 3101.251.25oligo3171.25oligo 3181.25PrimeStar Premix 2x25waterup to 50
  
ABC310 (F)LHRZ (BamHICCGGATCCCCGTGTAGGTCTACAAACTGG318 (R)LHRZ  (KpnI-BsmBI-AsiSI)GCGGTACCCGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG317 (R)LHRZ  (KpnI-BsaI-AsiSI)GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG
 

SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
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)
120 min  

  
components HLRZ (BsmBI)HLRZ (BsaI)eDA8 (125pg/uL)11oligo 3101.251.25oligo3171.25oligo 3181.25PrimeStar Premix 2x25waterup 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
 
ABC310 (F)LHRZ (BamHICCGGATCCCCGTGTAGGTCTACAAACTGG318 (R)LHRZ  (KpnI-BsmBI-AsiSI)GCGGTACCCGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG317 (R)LHRZ  (KpnI-BsaI-AsiSI)GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG
  
Programtimenumber of cycles98oC10 sec33 x55oC10sec68oC1min 30 sec4oChold
   
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1.2. 30 min 
PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit
Elution with water 50uL
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1.3. SalI and BamHI digestion of PCR product and pUC19 (all enzymes NEB)BamHI
  
  
 
 10 x CutSmart8888BamHI-HF (20u/uL)1.51.51.51.5eDA99 70 ng/uL30eDA101 81 ng/uL30PCR hygR 306/30750PCR zeo 308/30950SalI-HF (20u/uL)1.51.51.51.5waterup to 80 ->
          37 °C 240 min 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1.4. 30 min 
PCR clean-up QIAquick PCR Purification Kit (Qiagen)QIAquick PCR Purification Kit
Elution with water 50uL
  
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1.5. Ligation reactions T4 DNA ligase (NEB)

   
 T4 DNA ligase1110 X buffer11eDA101 (S/B)3zeo 308/309 (S/B)5eDA99 (S/B)3hygR 306/307 (S/B)5waterup to 10 ->
    


SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1.6. 120 min 

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB + 100ug/mL carbenicillin and growth at   37 °C 
  

SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1.7. 60 min 
.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   
(oligo 306/42  for verification HygR-LHRC-puC see plasmid map   
Material loaded on agarose gel for verification
 
 
Positively verified clones re-cultured in LB ampicillin for miniprep (plasmid preparation)   
SECTION: Cloning and preparation of HygR-LHRZ and ZeoR-LHRZ, integrative/insertion array lasting 3&#x27;-part
1.8. Plasmid isolation (miniprep) Qiagen miniprep 60 min 
PCR verified and Sanger sequencing intermediate plasmid ZeoR-LHRC-puC see plasmid   
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    The part is BsaI-free but contains internal AsiSI in hph gene , requires mutagenesis

 
2. Mutagenesis of AsiSI in eDA103 plasmid by Gibson assembly
 
 
  

2.1.  90 min PCR amplification for further Gibson assembly reaction
   
 PrimeStarHS 2x premix (Takara) High Fidelity
waterup to 50 ->Primestar2x2525oligos 327/3201.25/1.25oligos 319/3261.25/1.25eDA103 (74pg/uL)1.51.5
 
PROGRAM TAK-2.30   98C10sec33x55C10 sec68C2min 30 sec4Chold
   PCR reaction treatment with DpnI for 30 min at 37 °C 

2.2. 30 min   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
  
 30 min 37 °C 
Gibson1 319/326 (3.3kb) 40nM 3.3 kb1Gibson2 327/320 (2.05 kb) 40nM0.6Gibson 2x mix2.5water0.9
 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
60 min 
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   
 

2.7. Restriction digestion verification. Plasmid eDA115     digested with AsiSI (NEB)
  
 
 AsiSI 11NdeIeDA115 326 ng/uL3eDA103 103 ng/uL810 x CutSmart33waterup 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  

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

2.6. Plasmid isolation (miniprep) Qiagen miniprep 60 min