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    nanochromosome arrays combinatory assembly

    Dariusz Abramczyk, Dariusz Abramczyk

    Published: 2023-07-17 DOI: 10.17504/protocols.io.bp2l69p95lqe/v1
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    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 http://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'

    Steps

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

    1.

    DNA parts preparation by PCR reactions

    List of DNA parts

    ABCDE
    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 http://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

    1.1.

    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 http://doi.org/10.3389/fimmu.2021.681098
    Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

    volumes in microliter

    AB
    waterup to 50
    Q5 HF enzyme0.7
    eDA143 100pg/uL1
    betaine 5M10
    oligo447/ oligo4482.5/2.5
    10mM dNTP (TAKARA)4
    5xQ5 buffer10
    ABCD
    Td-initial98oC30sec
    Td98oC10sec33 cycles
    Ta55oC20sec
    Te72oC1 min 40sec  
    Final extension72oC2 min
    hold4oChold
    ABC
    F447PDI part (MfeI)CCCCAATTGACAAGCTTTTGATTTTAACG
    R448PDI part (NotI) introducing HIS-tag-TAA stop codon (italics)TTTTTGCGGCCGCTTAATGATGATGGTGGTGATGCAACTCATCGTGAGCATCAGCTTC
    PDI PCR product 1.7kb
    PDI PCR product 1.7kb

    PCR clean-up

    RE digested with MfeI/NotI

    pPICZA Thermo Fisher vectors

    ABC
    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 and incubation 0h 30m 0s

    37°C

    PCR product and pPICZA (MfeI/NotI) cleanup

    Ligation reactions T4 DNA ligase (NEB)

    AB
    pUC B/K BamHI/KpnI1
    T4 DNA ligase1
    Paox1PDI Mfe/NotI1
    10 X T4 buffer6
    waterup to 10 ->
    pPICZA MfeI/NotI1

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

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

    Several colonies obtained on both agar plates. Single colonies transferred on new LB +100ug/mL 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 in LB + 100 ug/ml carbenicillin, plasmid eDA226 was extracted by miniprep (plasmid preparation)

    1.2.

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

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

    volumes in microliter

    see oligo tables step 1 (above)

    ABCDE
    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
    waterup to 50 uLup to 50up to 50up to 50
    ABCD
    Td-initial98oC30sec
    Td98oC10sec33 cycles
    Ta58oC20sec
    Te72oC2min 30sec
    Final extension72oC1 min
    hold4oChold

    All PCR products purified by and analysed on 1% agarose gel (with)

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

    Digestion with or

    ABCDE
    LHRA 315/30146
    Paox1PDI 302/30346
    LHRE 336/33546
    HygR-LHRZ 306/31746
    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 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
    1.3.

    STEP 2 - preparation of semi-products, a partial ligated arrays

    ABC
    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 1h 0m 0s 25°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

    DNA concentration calculated by DeNovix DS-11

    ABC
    ng/uLMolar concentration (nM)
    LHRA-Paox1PDI104
    LHRE-HygR-LHRZ167
    1.4.

    pUC19 digestion with Esp3I

    Digestion with

    AB
    pUC19 500ng/uL5 uL
    Esp3I2 ul
    rCutsmart 10X4 ul
    waterup to 40 uL

    Digestion mixture cleanup and DNA concentration checked on DeNovix DS-11 and 1%agarose gel

    ABC
    ng/uLMolar concentration (nM)
    pUC19/Esp3I3519
    1.5.

    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
    LHRA-Paox1PDI (4nM)47
    LHRE-HygR-LHRZ (7nM) 74
    pUC19/Esp3I (19nM)191.5
    water-

    16h 0m 0s 16°C

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

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

    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

    Plasmids isolated by plasmid isolation (miniprep) Qiagen miniprep 1h 0m 0s

    Sanger sequencing confirmed the sequence of eDA227

    eda227 LHRA-PDI-LHRE-HYGR_LHRZ.dna

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

    2.

    DNA parts preparation by PCR reactions LHRE+PtefGFP (1st pre-assembly ligation mixture)

    List of DNA parts

    ABCDE
    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 http://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

    2.1.

    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

    volumes in microliter

    ABCD
    Q5 HF enzyme0.70.70.7
    10mM dNTP (TAKARA)444
    waterup to 50 uLup to 50up 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
    ABCD
    Td-initial98oC30sec
    Td98oC10sec33 cycles
    Ta58oC20sec
    Te72oC2min 10sec
    Final extension72oC1 min
    hold4oChold

    All PCR products purified by and analysed on 1% agarose gel (with)

    Digestion with or

    ABCD
    LHRE 335/33846
    PtefGFP 302a/304a46
    ZeoR-LHRZ 308/31846
    BsaI HF (20U/ul)2
    Esp3I (10U/ul))33
    buf10x cutsmart666
    water up to 60uLup to 60uLup to 60uL

    Reaction 37°C 6h 0m 0s

    All PCR products purified by 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
    2.2.

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

    AB
    expected size ~2.3kb
    2x T7 ligase buffer15
    LHRE (out)6.5
    PtefGFP7.5
    T7 DNA ligase1
    water-
    ul

    Ligation reaction 1h 0m 0s 25°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

    DNA concentration calculated by DeNovix DS-11

    ABCDE
    size kbpng/uLMolar concentration (nM)
    LHRA-PtefGDP~2.35537
    ZeoR-LHRZ*2.1710172
    2.3.

    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

    16h 0m 0s 16°C

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

    Selection on LB +100ug/mL carbanicillin + 50ug/mL zeocin

    growth at 37°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

    Next day a pasmid isolation (miniprep) Qiagen miniprep 1h 0m 0s

    Sanger sequencing confirmed the sequence of eDA197

    eda197 assembly.dna

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

    3.

    DNA parts preparation by PCR reactions

    List of DNA parts

    ABCDE
    DNA partForward oligoRevers oligoproducttemplate
    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 http://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

    3.1.

    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

    volumes in microliter

    AB
    Q5 HF enzyme0.7
    10mM dNTP (TAKARA)4
    waterup to 50
    oligo 302a/ oligo 304a2.5/2.5
    eDA191 100ng/ul1

    volume in microliters

    ABCD
    Td-initial98oC30sec
    Td98oC10sec33 cycles
    Ta58oC20sec
    Te72oC2min 10sec
    Final extension72oC1 min
    hold4oChold

    PCR product purified by and analysed on 1% agarose gel (with)

    Digestion with or

    AB
    Esp3I (10U/ul))3
    PtefMCh 302a/304a46
    buf10x cutsmart6
    water up to 60uL

    Reaction 37°C 6h 0m 0s

    All PCR products purified by 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
    3.2.

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

    AB
    ul
    expected size ~2.3kb
    PtefGFP7.5
    LHRE (out)6.5
    2x T7 ligase buffer15
    T7 DNA ligase1
    water-

    Ligation reaction (in duplicate) 1h 0m 0s 25°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

    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
    3.3.

    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-PtefMCH1.664
    ZeoR-LHRZ1.472
    pUC19/Esp3I519

    16h 0m 0s 16°C

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

    Selection on LB +100ug/mL carbanicillin + 150ug/mL hygromycin B

    growth at 37°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

    Next day a pasmid isolation (miniprep) Qiagen miniprep 1h 0m 0s

    Sanger sequencing confirmed the sequence of eDA199

    eda199 assembly.dna

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

    4.

    DNA parts preparation by PCR reactions

    List of DNA parts

    ABCDE
    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 http://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

    4.1.

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

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

    volumes in microliters

    ABCDE
    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
    waterup to 50 uLup to 50up to 50up to 50

    Part LHRE (out) generated as described above in protocol 2.1

    Part ZeoR-LHRZ generated as described above in protocol 2.1

    ABCD
    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 and analysed on 1% agarose gel (with)

    Digestion with or

    ABCDE
    LHRE 335/33846
    PaoxGFP:CFH 302/30346
    LHRD 304/30546
    ZeoR-LHRZ 308/31846
    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 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
    4.2.

    STEP 2 - preparation of semi-products, a partial ligated arrays

    ABC
    ZeoR-LHRZ-9
    LHRD-5
    water--
    T7 DNA ligase11
    2x T7 ligase buffer1515
    LHRE out5.8-
    PoaxGFP:CFH8.2-
    expected size ~6.9kbexpected size ~3.2kb
    ulul

    Ligation reaction 1h 0m 0s 25°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

    DNA concentration calculated by DeNovix DS-11

    ABC
    ng/uLMolar concentration (nM)
    LHRE-PaoxGFPCFH225
    LHRD-ZeoR-LHRZ94
    4.3.

    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-LHRZ46.2
    pUC19/Esp3I (19nM)191.3
    water

    16h 0m 0s 16°C

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

    Selection on LB +100ug/mL + 50ug/mL

    and growth at 37°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
    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

    Next day a pasmid isolation (miniprep) Qiagen miniprep 1h 0m 0s

    Sanger sequencing confirmed the sequence of eDA250

    puc19-E-PaoxGFPFH-D-ZeoC.dna

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

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