PLASMID ISOLATION AND ANALYSIS Part II Plasmid Purification and Isolation
Supplementary Figure 1 - NatureSupplementary Figure 4. Integration of 2 copies of BAD.øC31 gene....
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Supplementary Figure 1 BW27783 BW∆endA.KanR Figure 2 Knockout endA pBAD.Red fendA.attB.KanR.attP.endA BW∆endA Remove KanR p2øC31 Supplementary Figure 2 knockin TetR at lacY locus pBAD.Red, fz-TetR-a 3S2T.KanR 3S2T Remove KanR p2øC31 3S2T.KanR Supplementary Figure 4 Replace attR with KanR at ∆endA locus pBAD.Red, fendA.FRT.KanR.FRT.attB.endA Remove KanR pcI587.FLP 3S2T.attB 2P3S2T.KanR Integrate 2xBAD.øC31 p2øC31.attP.FRT, Inducing expression of øC31 integrase 2P3S2T Remove KanR pcI587.FLP Supplementary Figure 5 Integrate TetR at araD locus pBAD.Red, faraD.9attP.TetR.attB-araD 2P3S2T.TetR Integrate 4xBAD.øC31 Inducing expression of øC31 integrase 6P3S2T.TetR.KanR 6P3S2T Remove TetR.KanR pBAD.TPin, inducing expression of TPin integrase Supplementary Figure 6 Integrate TetR at galK locus pBAD.Red, fgalK. 9attP.TetR.attB- galK 6P3S2T.TetR Integrate 4xBAD.øC31 Inducing expression of øC31 integrase 10P3S2T.TetR.KanR ZYCY10P3S2T Remove TetR.KanR pBAD.TPin, inducing expression of TPin integrase BW∆endA.TetR 2T.KanR Replace TetR with lacY A177C pBAD.Red, fz-attB.KanR.attP.lacY A177C-a 2T Remove KanR p2øC31 Supplementary Figure 3 Knockin 3xBAD.I-SceI at UMU locus pBAD.Red, fUMU.araC3xBAD.I-SceI. attB.KanR.attP.UMU Supplementary Figure 1. A flowchart summarizing the stepwise genetic manipulation of the bacterial genome. The bacterial strains are boxed. Nature Biotechnology: doi: 10.1038/nbt.1708
Transcript of Supplementary Figure 1 - NatureSupplementary Figure 4. Integration of 2 copies of BAD.øC31 gene....
Supplementary Figure 1
BW27783 BW∆endA.KanR
Figure 2 Knockout endA pBAD.Red fendA.attB.KanR.attP.endA
BW∆endA Remove KanR p2øC31
Supplementary Figure 2 knockin TetR at lacY locus pBAD.Red, fz-TetR-a
3S2T.KanR 3S2T Remove KanR p2øC31
3S2T.KanR
Supplementary Figure 4 Replace attR with KanR at ∆endA locus pBAD.Red, fendA.FRT.KanR.FRT.attB.endA
Remove KanR pcI587.FLP
3S2T.attB 2P3S2T.KanR Integrate 2xBAD.øC31 p2øC31.attP.FRT, Inducing expression of øC31 integrase
2P3S2T Remove KanR pcI587.FLP
Supplementary Figure 5 Integrate TetR at araD locus pBAD.Red, faraD.9attP.TetR.attB-araD
2P3S2T.TetR Integrate 4xBAD.øC31 Inducing expression of øC31 integrase
6P3S2T.TetR.KanR 6P3S2T Remove TetR.KanR pBAD.TPin, inducing expression of TPin integrase
Supplementary Figure 6 Integrate TetR at galK locus pBAD.Red, fgalK. 9attP.TetR.attB-galK
6P3S2T.TetR Integrate 4xBAD.øC31 Inducing expression of øC31 integrase
10P3S2T.TetR.KanR ZYCY10P3S2T Remove TetR.KanR pBAD.TPin, inducing expression of TPin integrase
BW∆endA.TetR 2T.KanR Replace TetR with lacY A177C pBAD.Red, fz-attB.KanR.attP.lacY A177C-a
2T Remove KanR p2øC31
Supplementary Figure 3 Knockin 3xBAD.I-SceI at UMU locus pBAD.Red, fUMU.araC3xBAD.I-SceI. attB.KanR.attP.UMU
Supplementary Figure 1. A flowchart summarizing the stepwise genetic manipulation of the bacterial genome. The bacterial strains are boxed.
Nature Biotechnology: doi: 10.1038/nbt.1708
Ab-free Tet
Amp Kan
d!
b !Template TetRg BWg !
2.5-kb!
S!
Supplementary Figure 2!
Strain BW∆endA!
Strain BW∆endA.TetR!
pBAD.Red!
p2øC31!
Strains 2T!
c!
Pme I! Pme I!
Pbla.lacY A177C!
KanR!attP!attB!z! a!lacY A177C!
Pme1 !Restriction!
Integration !fragment!
+!
KanR!attP!attB!z! a!lacY A177C!Strain !2T.KanR!
attR!z! a!lacY A177C!
a!TetR!z!
AmpR! ColE1! F1!
KanR!attP!attB!z! a!lacY A177C!
pBAD.Red !
Strains BW∆endA.TetR!
a!
TetR!z! a!
Pme I! Pme1 restriction!
Integration fragment!
Pme I!
AmpR! ColE1!placY.TetR!
F1!
+!z! a!y!
TetR!z! a!
TetR!z! a!
Supplementary Figure 2. Integration of 2nd L-arabinose transporter mutant lacY A177C. (a) Flow chart illustrating procedure for preparing target site. placY.TetR, the plasmid used to prepare the Pme1-restricted integrating fragment; z, y and a, the structural genes of lactose operon; the boxed z and a, PCR-generated 425- and 227-bp of z- and a-specific sequences. (b) PCR illustration of integrant. 2.5-kb, integrant-specific PCR product; TetRg, genome of strain BW∆endA.TetR; BWg, genome of BW27783. (c) Mutant lacY A177C knock-in strategy. pbla.lacY A177C, the plasmid for producing the Pme1-restricted integrated DNA fragment; bla, the beta-galactosidase gene promoter. (d) Selection of the strain 2T. Colonies that lost all three antibiotic resistance phenotypes were selected using the 4 agar plates containing different antibiotics. Ab-free, antibiotic-free; Tet, tetracycline (12µg/ml); Amp, ampicillin (25 µg/ml); Kan, kanamycin (25 µg/ml).
Strain 3S2T Plasmid pBS.8I-SceIs Enzyme Xba I 1% L-arab - - + 37°C/hour 0 4 4
pBS.8I-SceIs!
ColE1!AmpR!
F1!
8I-SceIs!
b!
c!
a!
Strain 2T!
+!UMU!
pBAD.Red!
p2øC31!
Strain 3S2T!attR!UMU! UMU!araC.3BAD.I-SceI!
Pst I!
ColE1! F1!AmpR!
p3BAD.I-SceI!KanR! attP!attB!UMU! araC3xBAD.I-SceI! UMU!
Pst I!Pst I!Restriction!
Intermediate strain 3S2T.KanR !
Integrating DNA!KanR! attP!attB!UMU! araC3xBAD.I-SceI! UMU!
KanR! attP!attB!UMU! araC3xBAD.I-SceI! UMU!
Xba I
Supplementary Figure 3
Supplementary Figure 3. Integration of BAD.I-SceI gene. (a) Flow chart showing the BAD.I-SceI gene knock-in procedure. p3BAD.I-SceI, the plasmid encoding 3 tandem copies of the I-SceI gene under the control of BAD (BAD.I-SceI); UMU, the UMU D gene; the boxed UMU, PCR-generated 737- and 647-bp UMU-specific product, respetcively; araC, the repressor gene of araC.BDA system; integrating DNA, the DNA fragment derived from p3BAD.I-SceI via Pst 1 restriction; strain 2T, the E. coli expressing 2 constitutive L-arabinose transporters cp8.araE and bla.lacY A177C (Supplementary Figure 2); strain 3S2T, generated by Red-mediated integration of the above integrating fragment to the UMU gene of strain 2T. (b) pBS.8I-SceIs, a pBlueScript base plasmid encoding 8 tandem copies of the I-SceI site. (c) Demonstration of function of the integrated I-SceI gene in strain 3S2T. An overnight culture of the 3S2Tstrain transformed with pBS.8I-SceIs was re-suspended in LB with or without 1% L-arabinose and incubated at 37° for 4 hours; Xba I, the restriction enzyme used to cut the plasmid before electrophoresis.
Supplementary Figure 4!
e!
PP!
MC!
Parental pMC.RSV.hAAT!Enzyme Xba I+BamH I !%L-arab -- +!
a!
pBAD.Red !
pcI587.FLP!
+!
PCR!megaprimer! megaprimer!
Strain 3S2T!
endA! endA!FRT!
Strain 3S2T.attB!
KanR! FRT! attB!FRT!
pFRT.KanR.attB!
Integrating!DNA fragment!
strain 3S2T.KanR!
AmpR! F1!ColE1!
KanR! FRT! attB!FRT!endA!
attR! endA!endA!
endA endA
endA!
endA! KanR! FRT! attB!FRT! endA!
attB!
b!
+!
pcI587.FLP!
attR!endA! endA!FRT! araC! 2xBAD.øC31!
endA! endA! Strain 3S2T.attB!attB!
araC! 2xBAD.øC31! p2øC31.attP.FRT!
attP!FRT! KanR! R6K!Zeo!
attR!endA! endA!attL!FRT! FRT!KanR!R6K!Zeo! araC! 2xBAD.øC31!
Strain 2P3S2T!
strain 2P3S2T.KanR!
FRT!
ColE1!AmpR! 32I-SceIs!
pMC.RSV.hAAT!
attP!RSV.hAAT!attB!
d! BamHI Xba I Colony 1 2 !
7-kb!
c!
S!
Supplementary Figure 4. Integration of 2 copies of BAD.øC31 gene. (a) Preparation of genomic target site. pFRT.KanR.attB, the plasmid serving as PCR template; FRT, the binding site of flipase (FLP); megaprimers, 329- and 754-bp of endA sequences, generated by PCR, to serve as PCR primers for generating the integrating DNA fragment; the PCR product was integrated into the modified endA site of strain 3S2T via Red-mediated reaction; subsequently, the KanR was eliminated via FLP-mediated reaction between the two flanking FRTs, resulting in the strain 3S2T.attB; cI587, the bacteriophage Lambda temperature sensitive promoter. (b) Integration of BAD.øC31. p2øC31.attP.FRT, a conditional replicating plasmid carrying R6K origin capable of supporting plasmid replication only in the cell expressing the pir protein; Zeo, zeocin. (c) PCR illustration of the integrant. 7-kb, the size of the PCR product generated by a pair of endA-specific primers. S, partial DNA sequencing of the DNA in the band. (d) The minicircle producer plasmid pMC.RSV.HAAT. R, the Rous Sarcoma virus long terminal repeat (promoter); H, human alpha-1 antitrypsin cDNA; B, bovine growth hormone gene polyadenylation signal; 32I-SceIs, 32 consecutive I-SceI cutting sites. (e) Illustration of genomic øC31 integrase function. The minicircle was made according to the protocol described previously 6. Xba I+BamHI, 2 restriction enzymes used to cut the DNA before electrophoresis.
Ab-free Kan Tet!
c!
Clone ID 14 15 25 48 !
Integrant!S!
e!
Strain 6P3S2T!Plasmid pMC.RSV.hAAT!Enzyme Not I+BspEI!Reaction Overnight culture 50-ml, 1N NaOH 2.0-ml, variable volume of 20% Larabinose to final concentration of 0.01%!Fresh TB 0 Old prot 0 2.5 5.0 1 0 20 0 35 42.5 50 60-ml old prot!32°C/hr 0 2 5 2 5 2 5 2 5 2 5 2 5 0 2 5 2 5 2 5 2 5 2 5 !
f!
b !Template: BWg TetRg!
S!2.5-kb!
Kan+ Tet!
Supplementary Figure 5!
d!
a !
PCR!
pBAD.Red!
attB!9attP!araD! araD!
attB!TetR!9attP!araD! araD!
PolB!araD!araA!araB!araC!
TetR!
p4øC31.attP.9attB !
KanR! A101!
attP!
9attB!
araC! 4ØC31!+!
p9attP.TetR.attB!
araD ! araD!
TetR! attB!9attP!
megaprimer! megaprimer!Integration !fragment !
Strain 2P3S2T!
Strain !2P3S2T.TetR!
+!
F1!ColE1! AmpR!
pBAD.TPin!Strain 6P3S2T.KanR.TetR !
Strain 6P3S2T!
9attP! TetR! attL! KanR! 9attB!araC!4ØC31!attR! araD!A101!araD!
9attR!araC!4ØC31!attR! araD!araD!
Supplementary Figure 5. Integration of 4 copies of BAD.øC31. (a) Flow chart illustrating the integration procedure. p9attP.TetR.attB, the plasmid served as template for PCR-generation of the integrating DNA fragment; 9attB and 9attP, the bacterial and phage attachment sites of the bacteriophage TP901-1 integrase (TPin); the boxed araD, PCR-generated 275- and 310-bp araD-specific product, respectively; p4øC31.attP.9attB and pBAD.TPin, two complementing plasmids carrying the temperature-sensitive A101 origin of replication; 9attR, the recombination product between 9attB and 9attP. (b) PCR illustration of the altered araD locus. 2.5-kb, size of the PCR product generated by a TetR- and an araD-specific primers. (c) Selection of strain 6P3S2T.KanR.TetR. Kan+Tet, the plate containing both Kan and Tet. (d) Elimination of KanR and TetR. , marked colonies that lost both KanR and TetR are circled. (e) PCR illustration of integrant. Integrant, PCR product generated by a pair of araD-specific primers. (f) Optimizing minicircle production procedure. Reaction, minicircle formation reaction each comprised a 50-ml overnight culture, 2-ml 1N sodium hydroxide, indicated volume of fresh TB and L-arabinose to a final concentration of 0.01%; old prot, previous protocol for minicircle production 6.
Supplementary Figure 6!
c!
Ab-Free!
Kan!
Amp!
Tet!
10!15!
b !
Primer: galKup-+araC- øC31-+galKdn- !Template: 10 15 BW 10 15 BW!
d!
S!S!
PP2!PP1!
Template: Strain ! 6P3S2T.TetR! 1 2 3 4 BWg !
S!2.5-kb!
a !
PCR!
pBAD.Red!
attB!9attP!
galE!galT!galK!galM!
TetR!
p4øC31.attP.9attB!
KanR! A101!
attP!
9attB!
araC! 4ØC31!
galK! 9attP! TetR! attL! KanR! 9attB! araC! 4ØC31! attR! galK!A101!
pBAD.TPin!
galK! 9attR! araC! 4ØC31! attR! galK!
+!
galK ! galK!
p9attP.TetR.attB!TetR! attB!9attP!
AmpR! F1!ColE1!
megaprimer! megaprimer!
Integration !fragment !
Strain 2P3S2T!
Strain !6P3S2T.TetR!
Strain 6P3S2T.KanR.TetR!
Strain ZYCY10P3S2T!
attB!TetR!9attP!galK! galK!galE!galM!
+!
galK! galK!
Supplementary Figure 7. Empty Minicircle producer plasmid pMC.BESPX (1) DNA Strider map of ZYCY10P3S2T
Notes
• The sequence 1-730 encodes 32 consecutive cutting sites of homing endocnuclease I-SceI;
• The attB sequence “GGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCG” locates between 740-774 bp;
• The attP sequence “CCCAACTGGGGTAACCTTTGAGTTCTCTAGTTGGGG” locates between 825-860 bp;
• The following unique sites can be used to insert the transgene expression cassette of your interest: BglII, EcoRI, EcoRV, Sall, PspOMI and XbaI; pMC.BESPX carries neither promoter nor polyadenylation signal;
• It carries a Kanamycin resistance gene. (2) DNA sequence of pMC.BESPX ACATTACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGAtGACA TTACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCTAGATGACATT TACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACCCTGT TATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTACCCTGTTAT CCCTAGATACATTACCCTGTTATCCCAGATGACATACCCTGTTATCCCTA GATGACATTACCCTGTTATCCCAGATGACATTACCCTGTTATCCCTAGAT ACATTACCCTGTTATCCCAGATGACATACCCTGTTATCCCTAGATGACAT TACCCTGTTATCCCAGATGACATTACCCTGTTATCCCTAGATACATTACC CTGTTATCCCAGATGACATACCCTGTTATCCCTAGATGACATTACCCTGT TATCCCAGATGACATTACCCTGTTATCCCTAGATACATTACCCTGTTATC CCAGATGACATACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAG ATGACATTACCCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGA CATACCCTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATAAACTC
731 Not I •735 Sac I I764 Sma I764 Uth SI764 Xma I774 Spe I •780 Eco RI •786 Bgl II •787 Hae IV792 Eco RV •798 Xba I •804 Hin cII804 Sal I •815 EcoO109I815 Pss I816 Apa I816 PspOMI •833 Bst EII880 Acc 65I880 Kpn I901 Psi I907 Bsm I911 Not I •921 Ale I
1093 Rsr I I1109 Nae I1109 NgoMIV
1287 Fal I
1511 Fsp I1531 Msc I
1770 Bcl I1793 Xcm I
1933 Bts IBpm I 2066Xmn I 2095
Rle AI 2236Acl I 2382
Ppi I 2563
Afl III 3260Pci I 3260
CstMI 3406Nde I 3438Tat I 3454
Bst Z17I 3489Bsm BI 3611Pfo I 3615Bss HII 3683Bfr BI 3703Nsi I 3703
Ppu 10I 3703Mfe I 3772
Bcg I 3840Bcg I 3840
Dra I 3875
Bae I 3977Bae I 3977
Afl II 3999
pMC.BESPX
4084 bpUnique Sites
AATGATGATGATGATGATGGTCGAGACTCAGCGGCCGCGGTGCCAGGGCG TGCCCTTGGGCTCCCCGGGCGCGACTAGTGAATTCAGATCTGATATCTCT AGAGTCGACCCATGGGGGCCCGCCCCAACTGGGGTAACCTTTGAGTTCTC TCAGTTGGGGGTAATCAGCATCATGATGTGGTACCACATCATGATGCTGA TTATAAGAATGCGGCCGCCACACTCTAGTGGATCTCGAGTTAATAATTCA GAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAG CGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGC TCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGC CACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCA CCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCG CCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAG CCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCA TCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGG CAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGAT GGATACTTTCTCGGCAGGAGCAAGGTGTAGATGACATGGAGATCCTGCCC CGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGT CGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGC GCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGAC AAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAG AGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACC CAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAA CGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGA TCCTTGGCGGCGAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACC TTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAA AACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCT TTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACA TTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGCTCGA GgggGgccAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGAT TTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAAC AGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCG AACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCA TGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCG AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCT GAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGC CCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATT AAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCT TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGACCAAA ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCT TGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA GAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT ACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCA CACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAG CGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAG GTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTC CAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGC
CTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC CGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGAC CGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGT ATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACT CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCG CTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACC CGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC AAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCA TCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCG GCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCAAACCC TATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGAC AACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTC GCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTT GATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTG GTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCT TAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACG GCGACAAGCAAACATGCTGTGCGACGCTGGCGAT Supplementary Figure 7. Minicircle producer plasmid pMC.BESPX. DNA sequence and a DNA strider map are provided.
Supplementary Glossary Plasmid glossary
1. P2øC31-a plasmid expresses øC31 integrase under the control of the L-arabinose-
araC.BAD system; carrying a BAD.I-SceI cassette and an I-SceI site, the plasmid
can be eliminated through overnight culture with 1% L-arabinose.
2. p2øC31.attP.FRT- a plasmid for integrating 2 copies of BAD.øC31 cassette through
two FRT sites in the plasmid and in the genomic target, respectively; carrying the
conditional R6K DNA origin and capable of replicating only in the cell expressing the
pir protein, this plasmid will be cured in BW27783 and other common laboratory
strain lacking the pir gene.
3. p2øC31.hFIX, an old version of minicircle producer plasmid encoding the ApoE.hFIX
expression cassette, as described previously 2 . The makeup of the plasmid is
shown as p2øC31.Transgene when the expression cassette is the ApoE.hFIX
(ApoE HCR enhancer, hAAT promoter, hFIX minigene, and bovine poly A signal).
4. P2øC31.Transgene - the previous minicircle producer plasmid.
5. p3BAD.I-SceI -a plasmid for generating fragment DNA encoding 3 copies of BAD.I-
SceI cassette to be integrated through homologous recombination.
6. p4øC31.attP.9attP - a plasmid encoding 4 copies of BAD.øC31 cassette capable of
integrating through the øC31 integrase-mediated recombination between the attP
site in the plasmid and attB site in the genomic target.
7. p9attP.TetR.attB -a plasmid for integrating the TetR flanked with the attB and 9attP
sites into the genomic target site; 9attP, the plasmid attachment site of the
bacteriophage TP901-1 integrase (TPin).
8. pBAD.RED -a plasmid expressing the bacteriophage Lambda RED homologous
recombinase system under the control of the L-arabinose-araC.BAD system; control
under the heat sensitive DNA replication origin A101, it can be eliminated by growing
the cells at 43°C overnight.
9. pBAD.TPin - a plasmid expressing the TPin to mediate the elimination of unwanted
or harmful DNA elements through recombination between 9attB and 9attP, under
the control of the L-arabinose-araC.BAD system; similar to that of p2øC31, the
plasmid carrying the BAD.I-SceI cassette and an I-SceI site, allowing its own
degradation when inducing expression of I-SceI by L-arabinose.
10. pbla.LacY A177C -a plasmid for integrating the bla.LacY.A177C cassette at the
disrupted LacY region. Bla, promoter of beta-lactosidase gene; A177C, an Alanine to
Cysteine mutation at codon 177.
11. pBS.8I-SceIs - a pBlueScript base plasmid carrying 8 consecutive I-SceI sites.
12. pcI587.FLP - a plasmid expressing flipase, which mediates recombination between 2
FRT sites, under the control of bacteriophage Lambda pR promoter/cI587 repressor
system.
13. pFRT.KanR.attB -a plasmid for generating PCR product encoding an attB site and
the KanR flanked with 2 FRT sites up- and down-stream, respectively.
14. pKanR.endA - a plasmid for generating pme1-fragment to interrupt the endA gene.
15. pLacY.TetR - a plasmid for generating DNA fragment to disrupt the LacY locus with
the selection marker, TetR.
16. pMC.ApoE.hFIX -a new version of minicircle producer plasmid for producing
minicircle encoding the ApoE.hFIX expression cassette.
17. pMC.CMV.LGNSO - a new version of minicircle producing plasmid for production of
the minicircle encoding the reporter gfp and a set of 4 transcription factors, including
the LIN28, NANOG, SOX2 and OCT4 , for converting somatic cells to iPS cells
through reprogramming 1.
18. pMC.RSV.hAAT - a new version of minicircle producer plasmid for production of
minicircle encoding the RSV.hAAT expression cassette ( RSV-LTR promoter, hAAT
cDNA, and bovine poly A signal).
Supplementary Protocol: Minicircle production protocol Materials:
1. E. coli strain: ZYCY10P3S2T 2. Empty minicircle producing plasmid pMC.BESPX 3. Terrific Broth (TB) Powder (Invitrogen, Carlsbad, CA); 4. Luria-Bertani broth (LB-medium) powder (MP Biomedicals, Solon, OH); 5. Plasmid purification kits (Qiagen Volancia, CA).
Procedure:
1. Preparation of competent cell of ZYCY10P3S2T following standard protocol; 2. Transformation of the ZYCY10P3S2T competent cell with minicircle producing
plasmid carrying the transgene of interest following standard protocol; 3. Preparation of minicircle:
• On Day 1 morning, inoculate bacteria from 2 colonies in one each of 2-ml of LB containing kanamycin (50 µg/ml) at 37°C with shaking at 250 rpm;
• On Day 1 evening, prepare plasmid DNA and confirm the ID of the minicircle producing plasmid through restriction mapping;
• One Day 1 evening, cast an overnight culture by combining 100-µl of the culture from above step with 400-ml TB containing kanamycin (50 µg/ml) in 2 liter flask and incubate with shaking as in above step; if prepare smaller volume, keep the ratio of flask size to the TB volume at 5:1 (vol:vol) to ensure appropriate aeration;
• On Day 2 morning (culture for 16-18 hours), the OD600 reading of the overnight culture will be between 4 to 5, pH 6.5; otherwise, adjust to pH 6.5 using 1N Sodium Hydroxide;
• For a 400-ml overnight culture, prepare a Minicircle Induction Mix comprising 400-ml LB, 16-ml 1N NaOH and 400-µl 20% L-arabinose (final concentration in the Minicircle Induction Reaction will be 0.01%);
• Cast the Minicircle Induction Reaction by combine the Minicircle Induction Mix with the overnight culture and incubate at 32°C with shaking at 250 rpm for 5-hours; the incubation can be extended for additional 1 to 3 hours if desired;
• After pelleting the bacteria, use Qiagen kit to isolate minicircle according to manufacturer’s protocol with modifications: double the volume of P1, P2 and P3 buffers. For example, for 400-ml overnight culture, use 100-ml each of P1, P2, P3 buffers and one 2500 column;
• Note: to have the best yield, ensure that the bacteria are completely resuspended in P1 buffer; to minimize contamination of bacterial genomic DNA, avoid excessive shaking of the bacteria suspension after addition of P2 buffer.
