nanochromosome arrays combinatory assembly

Dariusz Abramczyk; Dariusz Abramczyk

Jul 16, 2023

nanochromosome arrays combinatory assembly

DOI

https://dx.doi.org/10.17504/protocols.io.bp2l69p95lqe/v1

Dariusz Abramczyk¹,
Dariusz Abramczyk¹

¹University of Edinburgh

Dariusz Abramczyk

University of Edinburgh

DOI: https://dx.doi.org/10.17504/protocols.io.bp2l69p95lqe/v1

Protocol Citation: Dariusz Abramczyk, Dariusz Abramczyk 2023. nanochromosome arrays combinatory assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69p95lqe/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. The protocol is a part of the project regarding the preparation of synthetic precursor of Pichia pastoris nanochromosomes.

Created: February 20, 2023

Last Modified: July 16, 2023

Protocol Integer ID: 77324

Keywords: nanochromosome array, nanochromosome, assembly the yeast pichia pastori, recombinant protein, yeast replication origin, assembled dna part, genetic engineering, expressing heterologous gene, mode of sequential gene integration, essential scaffolding features as telomere, dna arrays library, sequential gene integration, genome, value recombinant protein, genome in order, array of gene, heterologous gene, combinatory assembly, gene, dna sequence, yeast pichia pastori, modified protein, resistance gene, flanking oligonucleotide, dna part, molecular biology technique, chromosome, controlled insertion, dna band, insertion of multiple expression cassette, dna, replicating, t7 dna ligase, final ligation with linearlized puc adaptor plasmid, stable synthetic construct, linear dsdna suitable for insertion

Abstract

The yeast Pichia pastoris is used widely to biomanufacture high-value recombinant proteins. Its cells can secrete copious quantities of post-translationally modified proteins. Currently, however, heterologous genes must first be integrated into the genome in order to achieve expression.
We produced a synthetic 'chromosome-like' construct called here nanochromosome, an autonomously replicating and mitotically stable synthetic construct expressing heterologous genes. The nanochromosome contains essential scaffolding features as telomeres, centromere and yeast replication origins along with a versatile platform for genetic engineering. This platform could be used for the accurate and controlled insertion of multiple expression cassettes placed on 'landing pad',  in which an array of genes of interest alternate with ~1kp non-coding DNA sequences (LHR) chosen to facilitate simultaneous  double cross-overhomologous recombination and serve as spacers. The landing zone translates along the nanochromosome in an inchworming mode of sequential gene integrations that recycles a pair of antibiotic-resistance markers
 

  
Module with expression cassettes and LHR spacers as a pre-prepared in-vitro assembled DNA parts (insertion or integration arrays) is delivered to Pichia by transformation
Both the “integration” (receptor) and “insertion” (donor) arrays referred to herein consist of long, HR-ready, regions (LHRs) alternating with gene-expression cassettes. We constructed array parts containing either two or three LHRs e.g.LHRn-GoIn-AMRH/Z-LHRZ or LHRn-GoIn-LHRm-AMRH/Z-LHRZ, respectively, wherein: LHRn, LHRm and LHRZ are unique sequences of ~1000 bp from a library of LHRsA-Z, with LHRZ reserved as the last LHR in the array; GoIn  is a cassette consisting of a gene-of-interest with promoter and terminator regions; and AMRZ/H is a zeocin-resistance gene or hygromycin-resistance gene also with promoter and terminator regions. 
Preparation of ZeoR-LHRZ and HygR-LHRZ pair tandems in separate protocol https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

The protocol for multi-DNA parts arrays preparation is based on combinatory assembly and molecular biology techniques customised to create a DNA parts (arrays) library in pUC19 plasmid. 

Major 3 experimental steps:

Step 1 - DNA parts generated by PCR with a complementary overhangs designed to create a combined linear dsDNA suitable for insertion into BsmBI-sites of pUC19 (DNA arrays library)
- a part generating by PCR, oligos deliver a complementary overhangs for digestion with BsmBI (Esp3I) or BsaI, 
- PCR product digestion with BsmBI or BsaI
- checking digested PCR products on agarose gel
- PCR clean up OR gel extraction of DNA and clean up (only in the case if a PCR product is not homogenous 
- estimation of DNA concentration (molar)
Note: (*) stands for AsiSI-FspI RE recognition site provides in Fd-oligo1 and RV-olig4


  
Step 2 - preparation of semi-products before the the final ligation with linearlized pUC adaptor plasmid
-  ligation part1+2 with T7 DNA ligase
- ligation part 3+4 with T7 DNA ligase
- loading ligation mixtures and separation on agarose gel, 
- gel extraction of estimated DNA band
-  agarose gel verification, DNA concentration (molar)


Step3 - the final ligation of pre-assembled parts with BsmBI-linearized pUC19
- part1+2 and part 3+4 and linearized pUC19 ligation (DNA T7 ligase)
- transformation into E.coli and selection on LB ampicillin plates
- verification by bacterial colony PCR 
- plasmid isolation and Sanger (Plasmidsaurus) sequencing
- positively verified clone storage in glycerol stock 


The assembled part can be retained by:
- PCR amplification using flanking oligonucleotides
- Digested by AsiSI or FspI (need to be checked for the absence RE site in internal array) and digestion mixture can be used for Pichia transformation

Note: LHR part (e.g. LHRA) in flanking region (as a part 1) sometimes called 'OUT' while the LHRA in internal site (as a part 3) sometimes called 'IN'

Protocol materials

ReagentThiazole Orange Dye powderMerck MilliporeSigma (Sigma-Aldrich) 
ReagentBetain solution 5MMerck MilliporeSigma (Sigma-Aldrich)Catalog #B0300 
ReagentQIAquick PCR Purification KitQiagenCatalog #28104 
ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L 
ReagentBsaI-HF - 1,000 unitsNew England BiolabsCatalog #R3535S 
ReagentEsp3I - 300 unitsNew England BiolabsCatalog #R0734S 
ReagentNotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L 
ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g 
ReagentMfeI-HF - 500 unitsNew England BiolabsCatalog #R3589S 
ReagentAntarctic Phosphatase - 1,000 unitsNew England BiolabsCatalog #M0289S 
ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S 
ReagentQiagen Plasmid Plus Midi KitQiagenCatalog #12945 
ReagentPureLink&trade; Quick Gel Extraction KitThermo FisherCatalog #K210012 
ReagentHygromycin B, 50mg/ml solution (in PBS), sterileBio Basic Inc.Catalog #BS725.SIZE.2ml 
ReagentZeocinBio Basic Inc.Catalog #Z706211.SIZE.100mg 

Troubleshooting

Preparation of triple-LHR integration array LHRA-PAOXPDI-LHRE-HygR-LHRZ

30m

DNA parts preparation by PCR reactions
List of DNA parts 
DNA partForward oligoRevers oligo
producttemplate
LHRA (OUT) BsmBIF315-CCGTCTCCgggaGCGATCGCCTGTGTTAAACCGTCTTTAAGTCAACCCR301-GCGTCTCCtgtcCAAACTAAGATTGGACTATTGCTATC883 bpeDA8
PAOX1PDIhis(BsmBI)F302-CCGTCTCCgacaAACATCCAAAGACGAAAGGTTGR303-GCGTCTCCctgaTCTCACTTAATCTTCTGTACTCTG2905 bpeDA226*
LHRE (IN) (BsaI)F336-CGGTCTCCtcagGACTTACCTCGTTTTAACTTAGTCGGR350-GGGTCTCCctacACAAGTTTAACTAAGCGTCAGCC892 bpeDA9
HygR-LHRZ (BsaI)F306-CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCCR317-GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG2583 bpeDA115**
* see 1.1 for prepartion (below)
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

Prestep - Generation of pPICZA with POAX1PDIhis - (eDA226)
Plasmid pPICZalphaPDI eDA143 (reference citation)
Citation
Kerr H, Herbert AP, Makou E, Abramczyk D, Malik TH, Lomax-Browne H, Yang Y, Pappworth IY, Denton H, Richards A, Marchbank KJ, Pickering MC, Barlow PN (2021). Murine Factor H Co-Produced in Yeast With Protein Disulfide Isomerase Ameliorated C3 Dysregulation in Factor H-Deficient Mice. Frontiers in immunology.https://doi.org/10.3389/fimmu.2021.681098
LINK
 Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L  
ReagentBetain solution 5MMerck MilliporeSigma (Sigma-Aldrich)Catalog #B0300  
volumes in microliter
reagentsPDIhis
water up to 50
Q5 HF enzyme0.7
eDA143 100pg/uL1
betaine 5M10
oligo447/ oligo4482.5/2.5
10mM dNTP (TAKARA)4
5xQ5 buffer10
   
program Q5BCD
Td-initial98oC30sec
Td98oC10sec33 cycles
Ta55oC20sec
Te72oC1 min 40sec  
Final extension72oC2 min
hold4oChold
 
ABC
  F447  PDI  part (MfeI)  CCCCAATTGACAAGCTTTTGATTTTAACG  
  R448  PDI  part (NotI) introducing HIS-tag-TAA stop codon (italics)    TTTTTGCGGCCGCTTAATGATGATGGTGGTGATGCAACTCATCGTGAGCATCAGCTTC  
 
PDI PCR product 1.7kb
PCR clean-up ReagentQIAquick PCR Purification KitQiagenCatalog #28104   

RE digested with MfeI/NotI ReagentMfeI-HF - 500 unitsNew England BiolabsCatalog #R3589S  ReagentNotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L  
pPICZA Thermo Fisher vectors
 
reagentsPCR Paox1PDIpPICZA
12
pPICZA 100ng/uL13
PCR product of 447/44848
MfeI-Hf (20u/ul)1.51
NotI-Hf (20u/ul)1.51
rcutsmart86
waterup to 80up to 60
after digestion additional dephosorylation reation
+ 7ul AP buffer + 2uL Antarctic Phosphatase
 60 uL of pPICZA MfeI/NotI digested mixed with 7 ul AP buffer and 2 uL  ReagentAntarctic Phosphatase - 1,000 unitsNew England BiolabsCatalog #M0289S   and incubation Duration00:30:00   
Temperature37 °C   

PCR product and pPICZA (MfeI/NotI) cleanup  ReagentQIAquick PCR Purification KitQiagenCatalog #28104  

  Ligation reactions T4 DNA ligase (NEB)
 ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S  
 

A1
pUC B/K BamHI/KpnI1
T4 DNA ligase1
Paox1PDI Mfe/NotI 1
10 X T4 buffer6
waterup to 10 ->
pPICZA MfeI/NotI1
 DurationOvernight 16oC  

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

Several colonies obtained on both agar plates. Single colonies transferred on new  LB +100ug/mL ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g  plate, growth overnight and a single colonies submitted for bacterial colony PCR.
see protocol colony PCR
Material loaded on agarose gel for verification. 5 colonies verified positively using oligo 24/241.

 

 Positively verified clones re-cultured DurationOvernight    in LB + 100 ug/ml carbenicillin, plasmid eDA226 was extracted by miniprep (plasmid preparation) ReagentQiagen Plasmid Plus Midi KitQiagenCatalog #12945  

30m

STEP 1 - PCR amplification of parts and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L  
ReagentBetain solution 5MMerck MilliporeSigma (Sigma-Aldrich)Catalog #B0300  
volumes in microliter
 
see oligo tables step 1 (above)

reagentsLHRA
Paox1PDIhisLHREHygR-LHRZ
5xQ5 buffer10101010
betaine 5M10101010
10mM dNTP (TAKARA)4444
oligo 301/ oligo 3152.5/2.5
oligo 302/ oligo 3032.5/2.5
oligo 336/ oligo 3502.5/2.5
oligo 306/ oligo 3172.5/2.5
eDA8 100ng/ul1
eDA226 (see 1.1) 100ng/ul1
eDA9 100ng/ul1
eDa115 100ng/ul1
Q5 HF enzyme0.70.70.70.7
water  up to 50 uLup to 50 up to 50 up to 50 
 
program Q5BCD
Td-initial98oC30sec
Td98oC10sec33 cycles
Ta58oC20sec
Te72oC2min 30sec
Final extension72oC1 min
hold4oChold
  
All PCR products purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and analysed on 1% agarose gel (withReagentThiazole Orange Dye powderMerck MilliporeSigma (Sigma-Aldrich) ) 


 
size (bp)concentration (ng/ul)Molar concentration (nM)
LHRA8832848
Poax1PDI29056434
LHRE89282142
HygR-LHRZ258310160
 

 Digestion with ReagentBsaI-HF - 1,000 unitsNew England BiolabsCatalog #R3535S   or ReagentEsp3I - 300 unitsNew England BiolabsCatalog #R0734S  

 
reagentsLHRAPaox1PDIhis
LHREHygR-LHRZ
LHRA 315/30146
Paox1PDI 302/30346
LHRE 336/33546
HygR-LHRZ 306/317 46
BsaI HF (20U/ul)222
Esp3I (10U/ul))3
buf10x cutsmart6666
water up to 60uLup to 60uLup to 60uLup to 60uL
 All PCR products purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligation
 
ABCD
size (bp)concentration (ng/ul)Molar concentration (nM)
LHRA8832848
Poax1PDI29056434
LHRE89282142
HygR-LHRZ258310160
 

STEP 2 - preparation of semi-products, a partial ligated arrays 
 
reagentsLHRA-Paox1PDILHRE-HygR-LHRZ
expected size ~3.7kbexpected size ~3.47kb
2x T7 ligase buffer1515
HygR-LHRZ-10
LHRa5.7-
LHRE-4
Poax1PDI8.3-
T7 DNA ligase11
water--
ulul
Ligation reaction
 Duration01:00:00   Temperature25 °C   

All ligation mixtures loaded on 1% agarose gel and separated to excise a corresponding size band
 

 DNA bands representing  expected size were excised and purified using ReagentPureLink&trade; Quick Gel Extraction KitThermo FisherCatalog #K210012  

DNA concentration calculated by DeNovix DS-11
 
ABC
ng/uLMolar concentration (nM)
LHRA-Paox1PDI104
LHRE-HygR-LHRZ167
 

pUC19 digestion with Esp3I
 Digestion with ReagentEsp3I - 300 unitsNew England BiolabsCatalog #R0734S  

 
 
 
reagentsvector
pUC19 500ng/uL5 uL
Esp3I2 ul
rCutsmart 10X4 ul
waterup to 40 uL
 Digestion mixture cleanup ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and DNA concentration checked on DeNovix DS-11 and 1%agarose gel

ng/uLMolar concentration (nM)
pUC19/Esp3I3519
 

STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification

 ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S  
nMml
T4 DNA ligase1
10x T4 ligase buffer1.5
LHRA-Paox1PDI (4nM)47
LHRE-HygR-LHRZ (7nM) 74
pUC19/Esp3I (19nM)191.5
water-
 DurationOvernight   Temperature16 °C   

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

Several colonies obtained on both agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 150 ug/mL hygromycin B
ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g  ReagentHygromycin B, 50mg/ml solution (in PBS), sterileBio Basic Inc.Catalog #BS725.SIZE.2ml  

Growth overnight and a single colonies (8 colonies) submitted for bacterial colony PCR.
see protocol colony PCR
Material loaded on agarose gel for verification. 2 of 8 colonies verified positively using oligo 24/241. (expected 1.1kb amplicon) and 42/394 (0.91kb amplicon).

 
Colony 1 and 2 inoculated into LB + carbanicillin + hygromycin and growth DurationOvernight   
Plasmids isolated by plasmid isolation (miniprep) Qiagen miniprep Duration01:00:00   
Sanger sequencing confirmed the sequence of eDA227

eda227 LHRA-PDI-LHRE-HYGR_LHRZ.dna  

17h

Preparation of double-LHR integration array LHRE-PtefGFP-ZeoR-LHRZ

30m

DNA parts preparation by PCR reactions LHRE+PtefGFP (1st pre-assembly ligation mixture)
List of DNA parts 
 
 
 
DNA partForward oligoRevers oligo
producttemplate
LHRE (OUT) (BsaI)F335-CGGTCTCCgggaGCGATCGCGACTTACCTCGTTTTAACTTAGTCGGR338-GGGTCTCCtgtcACAAGTTTAACTAAGCGTCAGCC900 bpeDA9
PtefGFP (BsmBI)F302a-CCGTCTCCgacaAACATCCAAAGACGAAAGGTTGR304a-CCGTCTCCctacGCCTTCGAGCGTCCC1442 bpeDA189*
ZeoR-LHRZ (BsmBI)F308-CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCCR318-GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG2171 bpeDA105**
* eDA189 map .dna file eda189.dna  
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

STEP 1 - PCR amplification of parts LHRE(out)+PtefGFP and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L  
ReagentBetain solution 5MMerck MilliporeSigma (Sigma-Aldrich)Catalog #B0300  
volumes in microliter

reagentsLHRE (out)PtefGFPZeoR-LHRZ
Q5 HF enzyme0.70.70.7
10mM dNTP (TAKARA)444
water  up to 50 uLup to 50 up to 50
oligo 302a/ oligo 304a2.5/2.5
oligo 335/ oligo 3382.5/2.5
oligo 308/ oligo 3182.5/2.5
eDA189 100ng/ul1
eDA9 100ng/ul1
eDa105 100ng/ul1
 
program Q5BCD
Td-initial98oC30sec
Td98oC10sec33 cycles
Ta58oC20sec
Te72oC2min 10sec
Final extension72oC1 min
hold4oChold
  
All PCR products purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and analysed on 1% agarose gel (withReagentThiazole Orange Dye powderMerck MilliporeSigma (Sigma-Aldrich) ) 
  


 Digestion with ReagentBsaI-HF - 1,000 unitsNew England BiolabsCatalog #R3535S   or ReagentEsp3I - 300 unitsNew England BiolabsCatalog #R0734S  

reagentsLHREPtefGFPZeoR-LHRZ
LHRE 335/33846
PtefGFP 302a/304a46
ZeoR-LHRZ 308/318 46
BsaI HF (20U/ul)2
Esp3I (10U/ul))33
buf10x cutsmart666
water up to 60uLup to 60uLup to 60uL
 Reaction Temperature37 °C   Duration06:00:00   
All PCR products purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligation

  
 
ABCD
size (bp)concentration (ng/ul)Molar concentration (nM)
LHRE out90066113
PtefGFP14429298
ZeoR-LHRZ217111178
 
 

STEP 2 - preparation of semi-product, a partial ligated array LHRE-PtefGFP
 
 
 
reagentsLHRE-PtefGFP
expected size ~2.3kb
2x T7 ligase buffer15
LHRE (out)6.5
PtefGFP7.5
T7 DNA ligase1
water-
ul
Ligation reaction
 Duration01:00:00   Temperature25 °C   
 Ligation mixture loaded on 1% agarose gel and separated to excise a corresponding size band
 

DNA band representing  expected size was excised and purified using ReagentPureLink&trade; Quick Gel Extraction KitThermo FisherCatalog #K210012  

DNA concentration calculated by DeNovix DS-11 
 
ABCDE
size kbpng/uLMolar
  concentration (nM)
LHRA-PtefGDP~2.35537
ZeoR-LHRZ*2.1710172

STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification
 
 
ABC
uLnM
T4 DNA ligase1
10x T4 ligase buffer1
LHRE-PtefGFP2.337
ZeoR-LHRZ1.278
pUC19/Esp3I4.519
 
  DurationOvernight   Temperature16 °C   

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL carbanicillin + 50ug/mL zeocin
ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g  ReagentZeocinBio Basic Inc.Catalog #Z706211.SIZE.100mg   growth at DurationOvernight   Temperature37 °C   


Several fluorescent colonies obtained on both agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 50 ug/mL zeocin 
 

Single fluorescent colonies inoculated on fresh LB + carbanicillin/zeocin and growth DurationOvernight   
Next day a pasmid isolation (miniprep) Qiagen miniprep Duration01:00:00   
Sanger sequencing confirmed the sequence of eDA197
eda197 assembly.dna  
 

17h

Preparation of double-LHR insertion array LHRE-PtefMCH-HygR-LHRZ

 DNA parts preparation by PCR reactions
List of DNA parts 
ABCDE
DNA partForward oligoRevers oligo
producttemplate
LHRE (OUT) (BsaI)F335-CGGTCTCCgggaGCGATCGCGACTTACCTCGTTTTAACTTAGTCGGR338-GGGTCTCCtgtcACAAGTTTAACTAAGCGTCAGCC900 bpeDA9
PtefMCh (BsmBI)F302a-CCGTCTCCgacaAACATCCAAAGACGAAAGGTTGR304a-CCGTCTCCctacGCCTTCGAGCGTCCC1442 bpeDA191*
HygR-LHRZ (BsaI)F306-CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCCR317-GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG2583 bpeDA115**
 * eDA191 map .dna file eda191.dna  
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

STEP 1 - PCR amplification of parts 
LHRE(out) (done in section 2, step 2.1) with oligos 335/338
HygR-LHRZ (done in section 1, step 1.2) with oligos 306/317

PtefMCh amplification and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L  
ReagentBetain solution 5MMerck MilliporeSigma (Sigma-Aldrich)Catalog #B0300  
volumes in microliter
 
 
reagentsPtefMCh
Q5 HF enzyme0.7
10mM dNTP (TAKARA)4
water  up to 50 
oligo 302a/ oligo 304a2.5/2.5
eDA191 100ng/ul1
volume in microliters
 
program Q5BCD
Td-initial98oC30sec
Td98oC10sec33 cycles
Ta58oC20sec
Te72oC2min 10sec
Final extension72oC1 min
hold4oChold
  
PCR product purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and analysed on 1% agarose gel (withReagentThiazole Orange Dye powderMerck MilliporeSigma (Sigma-Aldrich) ) 
 

 
 Digestion with ReagentBsaI-HF - 1,000 unitsNew England BiolabsCatalog #R3535S   or ReagentEsp3I - 300 unitsNew England BiolabsCatalog #R0734S  

 
 
 
reagentsTtefMCh
Esp3I (10U/ul))3
PtefMCh 302a/304a46
buf10x cutsmart6
water up to 60uL
 Reaction Temperature37 °C   Duration06:00:00   
All PCR products purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligation

  
ABCD
size (bp)concentration (ng/ul)Molar
  concentration (nM)
LHRE out90066113
PtefMCh14429298
HygR-LHRZ258310160

STEP 2 - preparation of semi-product, a partial ligated array LHRE-PtefMCh
 

 
reagentsLHRE-PtefMCh
ul
expected size ~2.3kb
PtefGFP7.5
LHRE (out)6.5
2x T7 ligase buffer15
T7 DNA ligase1
water-
Ligation reaction (in duplicate)
 Duration01:00:00   Temperature25 °C   
 Ligation mixture loaded on 1% agarose gel and separated to excise a corresponding size band
 

DNA band representing  expected size was excised and purified using ReagentPureLink&trade; Quick Gel Extraction KitThermo FisherCatalog #K210012  

DNA concentration calculated by DeNovix DS-11 
 
 
ABCD
size kbpng/uLMolar concentration (nM)
LHRE-PtefMCH~2.39664
HygR-LHRZ*2.510172
 * HygR-LHRZ from step 1.2 

STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification
 
uLnM
T4 DNA ligase1
10x T4 ligase buffer1
LHRE-PtefMCH1.664
ZeoR-LHRZ1.472
pUC19/Esp3I519
 
  DurationOvernight   Temperature16 °C   

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL carbanicillin + 150ug/mL hygromycin B
ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g  
ReagentHygromycin B, 50mg/ml solution (in PBS), sterileBio Basic Inc.Catalog #BS725.SIZE.2ml  
growth at DurationOvernight   Temperature37 °C   

Several fluorescent colonies obtained on both LB agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 50 ug/mL zeocin 
 

Single fluorescent colonies inoculated on fresh LB + carbanicillin/hygromycin and growth DurationOvernight   
Next day a pasmid isolation (miniprep) Qiagen miniprep Duration01:00:00   
Sanger sequencing confirmed the sequence of eDA199
eda199 assembly.dna  

17h

Preparation of triple-LHR insertion array LHRA-GFP:CFH-LHRE-ZeoR-LHRZ

DNA parts preparation by PCR reactions
List of DNA parts 
 
DNA partForward oligoRevers oligo
producttemplate
LHRE (OUT) (BsaI)F335-CGGTCTCCgggaGCGATCGCGACTTACCTCGTTTTAACTTAGTCGGR338-GGGTCTCCtgtcACAAGTTTAACTAAGCGTCAGCC900 bpeDA9
PAOX1GFP:CFH (BsmBI)F302-CCGTCTCCgacaAACATCCAAAGACGAAAGGTTGR303-GCGTCTCCctgaTCTCACTTAATCTTCTGTACTCTG5991bpeDA89*
LHRD (IN) (BsmBI)F304-CCGTCTCCtcagGACAGCAACCTAACCGACR305-GCGTCTCCctacCAGTCCTCGTGAAAGACGAG1105 bpeDA9
ZeoR-LHRZ (BsmBI)F308-CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCCR318-GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG2171 bpeDA105**
* map eDA89.dna   plasmid prepared as described in the paper (DOI.....PBarlow,DAbramczyk)
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

STEP 1 - PCR amplification of parts and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L  
ReagentBetain solution 5MMerck MilliporeSigma (Sigma-Aldrich)Catalog #B0300  
volumes in microliters
 

 
reagentsLHRE (out)Paox1GFP:CFHLHRD (in)ZeoR-LHRZ
5xQ5 buffer10101010
betaine 5M10101010
10mM dNTP (TAKARA)4444
oligo 335/ oligo 3382.5/2.5
oligo 302/ oligo 3032.5/2.5
oligo 304/ oligo 3052.5/2.5
oligo 308/ oligo 3182.5/2.5
eDA89 100ng/uL1
eDA9 100ng/ul11
eDa105 100ng/ul1
Q5 HF enzyme0.70.70.70.7
water  up to 50 uLup to 50 up to 50 up to 50 
 Part LHRE (out) generated as described above in protocol 2.1
Part ZeoR-LHRZ generated as described above in protocol 2.1

program Q5BCD
Td-initial98oC30sec
Td98oC10sec33 cycles
Ta58oC20sec
Te72oC*1min 10 sec (for LHRD) ** 5min 30 sec for Poax1GFP:CFH
Final extension72oC1 min
hold4oChold
  
All PCR products purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and analysed on 1% agarose gel (withReagentThiazole Orange Dye powderMerck MilliporeSigma (Sigma-Aldrich) ) 
 
 

  

 Digestion with ReagentBsaI-HF - 1,000 unitsNew England BiolabsCatalog #R3535S   or ReagentEsp3I - 300 unitsNew England BiolabsCatalog #R0734S  

 
 
 
reagentsLHREPaoxGFP:CFHLHRDZeoR-LHRZ
LHRE 335/33846
PaoxGFP:CFH 302/30346
LHRD 304/30546
ZeoR-LHRZ 308/318 46
BsaI HF (20U/ul)2
Esp3I (10U/ul))333
buf10x cutsmart6666
water up to 60uLup to 60uLup to 60uLup to 60uL
 All PCR products purified by ReagentQIAquick PCR Purification KitQiagenCatalog #28104  and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligati

ABCD
size (bp)concentration (ng/ul)Molar concentration (nM)
LHRE out9006648
PaoxGFPCFH599112334
LHRD1105120142
ZeoR-LHRZ217111178
  

STEP 2 - preparation of semi-products, a partial ligated arrays 
 
 
 
reagentsLHRE-Paox1GFP:CFHLHRD-ZeoR-LHRZ
ZeoR-LHRZ-9
LHRD-5
water--
T7 DNA ligase11
2x T7 ligase buffer1515
LHRE out5.8-
PoaxGFP:CFH 8.2-
expected size ~6.9kbexpected size ~3.2kb
ulul
Ligation reaction
 Duration01:00:00   Temperature25 °C   

All ligation mixtures loaded on 1% agarose gel and separated to excise a corresponding size band

 

DNA band representing  expected size was excised and purified using ReagentPureLink&trade; Quick Gel Extraction KitThermo FisherCatalog #K210012  

DNA concentration calculated by DeNovix DS-11 
  
   
ng/uLMolar concentration (nM)
LHRE-PaoxGFPCFH225
LHRD-ZeoR-LHRZ94
 

STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification

 
ABC
nMml
T4 DNA ligase1
10x T4 ligase buffer1.5
LHRE-PaoxGFPCFH55.0
LHRD-ZeoR-LHRZ 46.2
pUC19/Esp3I (19nM)191.3
water
  DurationOvernight   Temperature16 °C   

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL + 50ug/mL
ReagentCarbenicillin, disodium saltBio Basic Inc.Catalog #CDJ469.SIZE.1g 
ReagentZeocinBio Basic Inc.Catalog #Z706211.SIZE.100mg  

 and growth at DurationOvernight   Temperature37 °C   



Note
No fluorescence observed, possibly not GFP leaking on yeast Paox1 promoter in E.coli
  

Several colonies obtained on both agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 50 ug/mL zeocin B.
Two of them submitted for bacterial colony PCR.
see protocol colony PCR
 
ABCDEFGH
352/353274/278355/354345/40351/133374/370 
puc-E 0.45kbGFP-FH 0.75kb Aox-D 0.29kbD-zeo 0.9kbzeoc-C 0.6kb0.7kb contr+
1clone1 (eDA250)OKOKOKOKOKOK
2clone2nonenonepoornonenonenone
 
oligos positions on plasmid map in the file 
Material loaded on agarose gel for verification. 1 of 2 colonies verified positively verified

The colony inoculated on fresh LB + carbanicillin/zeocin and growth DurationOvernight   
Next day a pasmid isolation (miniprep) Qiagen miniprep Duration01:00:00   
Sanger sequencing confirmed the sequence of eDA250
puc19-E-PaoxGFPFH-D-ZeoC.dna  

Plasmidsaurus sequencing confirms the correct sequence (added to .dna file)

17h

DNA part	Forward oligo	Revers oligo	product	template
LHRA (OUT) BsmBI	F315-CCGTCTCCgggaGCGATCGCCTGTGTTAAACCGTCTTTAAGTCAACCC	R301-GCGTCTCCtgtcCAAACTAAGATTGGACTATTGCTATC	883 bp	eDA8
PAOX1PDIhis(BsmBI)	F302-CCGTCTCCgacaAACATCCAAAGACGAAAGGTTG	R303-GCGTCTCCctgaTCTCACTTAATCTTCTGTACTCTG	2905 bp	eDA226*
LHRE (IN) (BsaI)	F336-CGGTCTCCtcagGACTTACCTCGTTTTAACTTAGTCGG	R350-GGGTCTCCctacACAAGTTTAACTAAGCGTCAGCC	892 bp	eDA9
HygR-LHRZ (BsaI)	F306-CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCC	R317-GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG	2583 bp	eDA115**

	reagents	PDIhis
	water	up to 50
	Q5 HF enzyme	0.7
	eDA143 100pg/uL	1
	betaine 5M	10
	oligo447/ oligo448	2.5/2.5
	10mM dNTP (TAKARA)	4
	5xQ5 buffer	10

program Q5	B	C	D


Td-initial	98oC	30sec
Td	98oC	10sec	33 cycles
Ta	55oC	20sec
Te	72oC	1 min 40sec
Final extension	72oC	2 min
hold	4oC	hold

A	B	C
F447	PDI part (MfeI)	CCCCAATTGACAAGCTTTTGATTTTAACG
R448	PDI part (NotI) introducing HIS-tag-TAA stop codon (italics)	TTTTTGCGGCCGCTTAATGATGATGGTGGTGATGCAACTCATCGTGAGCATCAGCTTC

reagents	PCR Paox1PDI	pPICZA
1	2
pPICZA 100ng/uL		13
PCR product of 447/448	48
MfeI-Hf (20u/ul)	1.5	1
NotI-Hf (20u/ul)	1.5	1
rcutsmart	8	6
water	up to 80	up to 60
		after digestion additional dephosorylation reation
		+ 7ul AP buffer + 2uL Antarctic Phosphatase

	A	1
	pUC B/K BamHI/KpnI	1
	T4 DNA ligase	1
	Paox1PDI Mfe/NotI	1
	10 X T4 buffer	6
	water	up to 10 ->
	pPICZA MfeI/NotI	1

reagents	LHRA	Paox1PDIhis	LHRE	HygR-LHRZ
5xQ5 buffer	10	10	10	10
betaine 5M	10	10	10	10
10mM dNTP (TAKARA)	4	4	4	4
oligo 301/ oligo 315	2.5/2.5
oligo 302/ oligo 303		2.5/2.5
oligo 336/ oligo 350			2.5/2.5
oligo 306/ oligo 317				2.5/2.5
eDA8 100ng/ul	1
eDA226 (see 1.1) 100ng/ul		1
eDA9 100ng/ul			1
eDa115 100ng/ul				1
Q5 HF enzyme	0.7	0.7	0.7	0.7
water	up to 50 uL	up to 50	up to 50	up to 50


	size (bp)	concentration (ng/ul)	Molar concentration (nM)
LHRA	883	28	48
Poax1PDI	2905	64	34
LHRE	892	82	142
HygR-LHRZ	2583	101	60

reagents	LHRA-Paox1PDI	LHRE-HygR-LHRZ
	expected size ~3.7kb	expected size ~3.47kb
2x T7 ligase buffer	15	15
HygR-LHRZ	-	10
LHRa	5.7	-
LHRE	-	4
Poax1PDI	8.3	-
T7 DNA ligase	1	1
water	-	-
	ul	ul

A	B	C
	ng/uL	Molar concentration (nM)
LHRA-Paox1PDI	10	4
LHRE-HygR-LHRZ	16	7

	reagents	vector
	pUC19 500ng/uL	5 uL
	Esp3I	2 ul
	rCutsmart 10X	4 ul
	water	up to 40 uL


	nM	ml
T4 DNA ligase		1
10x T4 ligase buffer		1.5
LHRA-Paox1PDI (4nM)	4	7
LHRE-HygR-LHRZ (7nM)	7	4
pUC19/Esp3I (19nM)	19	1.5
water		-

reagents	LHRE (out)	PtefGFP	ZeoR-LHRZ
Q5 HF enzyme	0.7	0.7	0.7
10mM dNTP (TAKARA)	4	4	4
water	up to 50 uL	up to 50	up to 50
oligo 302a/ oligo 304a		2.5/2.5
oligo 335/ oligo 338	2.5/2.5
oligo 308/ oligo 318			2.5/2.5
eDA189 100ng/ul		1
eDA9 100ng/ul	1
eDa105 100ng/ul			1

	reagents	LHRE-PtefGFP
		expected size ~2.3kb
	2x T7 ligase buffer	15
	LHRE (out)	6.5
	PtefGFP	7.5
	T7 DNA ligase	1
	water	-
		ul

A	B	C	D	E
	size kbp	ng/uL	Molar concentration (nM)
LHRA-PtefGDP	~2.3	55	37
ZeoR-LHRZ*	2.17	101	72

	reagents	PtefMCh
	Q5 HF enzyme	0.7
	10mM dNTP (TAKARA)	4
	water	up to 50
	oligo 302a/ oligo 304a	2.5/2.5
	eDA191 100ng/ul	1

	reagents	TtefMCh
	Esp3I (10U/ul))	3
	PtefMCh 302a/304a	46
	buf10x cutsmart	6
	water	up to 60uL

	reagents	LHRE-PtefMCh
		ul
		expected size ~2.3kb
	PtefGFP	7.5
	LHRE (out)	6.5
	2x T7 ligase buffer	15
	T7 DNA ligase	1
	water	-

reagents	LHRE (out)	Paox1GFP:CFH	LHRD (in)	ZeoR-LHRZ
5xQ5 buffer	10	10	10	10
betaine 5M	10	10	10	10
10mM dNTP (TAKARA)	4	4	4	4
oligo 335/ oligo 338	2.5/2.5
oligo 302/ oligo 303		2.5/2.5
oligo 304/ oligo 305			2.5/2.5
oligo 308/ oligo 318				2.5/2.5
eDA89 100ng/uL		1
eDA9 100ng/ul	1		1
eDa105 100ng/ul				1
Q5 HF enzyme	0.7	0.7	0.7	0.7
water	up to 50 uL	up to 50	up to 50	up to 50

reagents	LHRE	PaoxGFP:CFH	LHRD	ZeoR-LHRZ
LHRE 335/338	46
PaoxGFP:CFH 302/303		46
LHRD 304/305			46
ZeoR-LHRZ 308/318				46
BsaI HF (20U/ul)	2
Esp3I (10U/ul))		3	3	3
buf10x cutsmart	6	6	6	6
water	up to 60uL	up to 60uL	up to 60uL	up to 60uL

Public workspacenanochromosome arrays combinatory assembly

nanochromosome arrays combinatory assembly

reagents	LHRE-Paox1GFP:CFH	LHRD-ZeoR-LHRZ
ZeoR-LHRZ	-	9
LHRD	-	5
water	-	-
T7 DNA ligase	1	1
2x T7 ligase buffer	15	15
LHRE out	5.8	-
PoaxGFP:CFH	8.2	-
	expected size ~6.9kb	expected size ~3.2kb
	ul	ul


	ng/uL	Molar concentration (nM)
LHRE-PaoxGFPCFH	22	5
LHRD-ZeoR-LHRZ	9	4

A	B	C	D	E	F	G	H
	352/353	274/278	355/354	345/40	351/133	374/370
		puc-E 0.45kb	GFP-FH 0.75kb	Aox-D 0.29kb	D-zeo 0.9kb	zeoc-C 0.6kb	0.7kb contr+
1	clone1 (eDA250)	OK	OK	OK	OK	OK	OK
2	clone2	none	none	poor	none	none	none