Paris/Freezer

From 2007.igem.org

(Difference between revisions)
(Strains (glycerol stock))
(Strains (glycerol stock))
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|<bbpart>pSB1A2</bbpart>::<bbpart>BBa_J37028</bbpart>+<bbpart>BBa_E0840</bbpart>
|<bbpart>pSB1A2</bbpart>::<bbpart>BBa_J37028</bbpart>+<bbpart>BBa_E0840</bbpart>
|Lox71-gfp tri-part (L12)  
|Lox71-gfp tri-part (L12)  
-
|L12.1 ok
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|L12.2 ok
|?
|?
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   

Revision as of 15:35, 15 August 2007

Contents

PCR products

PCR products
Number Name Oligo F Oligo R Matrix Length Comments Product Left ? []ng/µL Date
P1 Lox71-FtsA-FtsZ-1 O3 Lox71-FtsA-F O4 DEcoR1-FtsZ-R MG1655 1537 Ø
P2 FtsZ-2 O5 DEcoR1-FtsZ-F O2 FtsZ-R MG1655 1088 Ø
P3 DGAT-1
P4 DGAT-2
P5 Lox66-DapAColi O6 Lox66-DapAColi-F O7 DapAColi-R MG1655 985 Ø July 17, July 30
P6 Lox66-DapASubtilis O8 Lox66-DapASubt-F O9 DapASubt-R B.Subtilis 168 979 July 17, July 27, July 30
P7 Lox71-FtsZ O1 Lox71-FtsZ-F O2 FtsZ-R MG1655 1260 EcoRI site in FtsZ not mutated July 17, July 30
P8 RBS-DapAColi O20 RBS-DapAColi-F O7 DapAColi-R MG1655 952 B0030-DapAColi July 23, July 30
P9 RBS-DapASubtilis O21 RBS-DapASubt-F O9 DapASubt-R B.Subtilis 168 946 B0030-DapASubtilis July 23, July 27
P10 Lox71-FtsA-FtsZ O3 Lox71-FtsA-F O2 FtsZ-R MG1655 2580 EcoRI site in FtsZ not mutated July 23
P11 Lox71-KmR-Lox66 O10 Lox71-KmR-F O11 Lox66-KmR-R MP12 1084 July 27, July 30

http://partsregistry.org/images/partbypart/icon_terminator.png http://partsregistry.org/images/partbypart/icon_regulatory.png http://partsregistry.org/images/partbypart/icon_rbs.png http://partsregistry.org/images/partbypart/icon_reporter.png http://partsregistry.org/wiki/images/7/7b/Part_icon_cds.png http://partsregistry.org/wiki/images/9/9f/Part_icon_dna.png http://partsregistry.org/wiki/images/2/2e/Part_icon_generator.png http://partsregistry.org/wiki/images/a/ab/Part_icon_plasmid.png

Plasmids: MiniPrep Products

Plasmids: MiniPrep Products
Number Name Description Products in use Date
MP1 pJ23100 in J61002 Strong constitutive promoter in J61002 (clones 1 + 2) July 15
MP2 pJ23107 in J61002 Medium constitutive promoter in J61002 (clones 1 + 2) MP2.2 July 15
MP3 B0015 in pSB1AK3 Double terminator (clones 1 + 2) MP3.1 July 16
MP4 I0500 in pSB2K3 AraC pBAD (clones 1 + 2) July 16
MP5 BBa_B0030 in pSB1A2 Strong RBS (clones 1 + 2) July 18
MP6 BBa_E0422 in pSB1A2 ECFP (RBS+LVA+Term) (clones 1 + 2) MP6'.1, MP6'.2 July 18, July 24
MP7 BBa_E0241 in pSB1A2 PoPs to GFP converter (clones 1 + 2) MP7'.1 July 18, July 24
MP8 BBa_E0840 in pSB1A2 gfp tri-part; strong rbs (clones 1 + 2) July 18
MP9 BBa_J61047 in pSB1A2 Cre ORF (clones 1 + 2) MP9'.1, MP9'.2 July 18, July 24
MP10 BBa_I0500 in pSB2K3 AraC pBAD (clones 1 + 2) July 20
MP11 BBa_pJ23107 in p61002 Medium constitutive promoter in J61002 (clones 3 + 4) MP11'.3 July 20, July 23
MP12 BBa_P1003 in pSB1A2 KanR July 27
L1 BBa_I0500 in J61002 construct with pBad promotor regulating expression of mRFP L1".1, L1".2 July 25, July 27, Aug 7
L2 Lox66DapAE.coli in pSB1A2 Insertion of lox66DapAE.coli construct as a biobrick in pSB1A2 plasmid

http://partsregistry.org/wiki/images/9/9f/Part_icon_dna.png http://partsregistry.org/wiki/images/7/7b/Part_icon_cds.png

July 25
L6 BBa_B0030+BBa_J61047 strong RBS(B0030)-Cre ORF

http://partsregistry.org/images/partbypart/icon_rbs.png http://partsregistry.org/wiki/images/7/7b/Part_icon_cds.png

L6.1, L6.2, L6.3, L6".1, L6".2 July 25, July 27
L9 lox66 in pSB1A2
L10 lox71 in pSB1A2
L12 lox71-gfp in pSB1A2 construction of the biobrick lox71-gfp

http://partsregistry.org/wiki/images/9/9f/Part_icon_dna.png http://partsregistry.org/images/partbypart/icon_reporter.png

L12.1 & L12.2 July 30
L25 lox71-KmR-lox66 in pSB1A2 ligation of lox71-KmR-lox66 as a biobrick in pSB1A2 plasmid

http://partsregistry.org/wiki/images/9/9f/Part_icon_dna.png http://partsregistry.org/wiki/images/2/2e/Part_icon_generator.png http://partsregistry.org/wiki/images/9/9f/Part_icon_dna.png

L25.1 July 30
MPT1 pTOPO P5 MPT1.1, MPT1.2
MPT3 pTOPO P6 MPT3.2, MPT3.1 Aug 7
MPT4 pTOPO P8 MPT4.1, .2, .3, .4& .5 Aug 7
MPT5 pTOPO P9 MPT5.1, .2 &.3 Aug 7

Digestion Products


PURPLE = Vector
GREEN = Insert

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description Product Left []ng/µL Date
D1 pJ23100 pJ23100 Spe1 Pst1 2096 Vector with the J23100 promoter ready for insertion of a RBS/CDS insert 34 July 17
D2 mRFP pJ23100 Spe1 Pst1 887 27 July 17
D3 pJ23107 pJ23107 Spe1 Pst1 2096 Vector with the J23107 promoter ready for insertion of a RBS/CDS insert 32 July 17
D4 mRFP pJ23107 Spe1 Pst1 887 25 July 17
D5 pJ23100 pJ23100 EcoR1 Spe1 2925 The vector with the mRFP gene ready for insertion of a promoter. We are not sure the digestion worked properly. 28&29 July 17
D6 pJ23107 pJ23107 EcoR1 Spe1 2925 The vector with the mRFP gene ready for insertion of a promoter. We are not sure the digestion worked properly. 31&27 July 17
D7 Lox66 DapA E.coli Lox66 DapA E.coli (PCR product) EcoR1 Pst1 977 24 July 17
D8 Lox66 DapA E.coli Lox66 DapA E.coli (PCR product) Xba1 Pst1 962 44 July 17
D9 pJ23100 ES vector pJ23100 (MP1.1 & MP1.2) EcoI SpeI 2925 Vector with mRFP RBS/CDS ready for insertion promoter (fw-insert) 80&43 July 21
D10 pJ23100 promoter fw-insert pJ23100 (MP1.1 & MP1.2) EcoI SpeI 58 J23100 promoter ready for use as a fw-insert 35&28 July 21
D11 pJ23107 ES vector pJ23107 (MP2.1, MP2.2, MP2.3 & MP2.4) EcoRI SpeI 2925 Vector with mRFP RBS/CDS ready for insertion of a fw-insert promoter 49,35,28&41 July 21
D12 pJ23107 promoter fw-insert pJ23107 (MP2.1, MP2.2, MP2.3 & MP2.4) EcoRI SpeI 58 J23107 promoter ready for usage as a fw-insert 23,25,32&32 July 21
D13 mRFP pJ23107 (MP2.2, MP2.3 & MP2.4) SpeI PstI 887 mRFP RBS/CDS/T sequence, can it be used as a backward insert? => yes but you will not be able to add any biobrick after it 21&41 July 21
D14 Cre ORF J61047 MP9.1 & MP9.2 XbaI PstI 1063 Cre ORF extracted for usage as a bw-insert Ø D14.1 38&41 July 21
D15 B0030 bw-vector B0030 MP5.1 & MP5.2 SpeI PstI 2076 bacward vector for cloning a CDS bw-insert after the B0030 RBS 87&42 July 21
D18 BB dig. lox66DapAE.coli lox66-DapAE.coli P5 PCR product EcoRI PstI ~960 PCR product digested for cloning as a BioBrick Ø 44 July 21
D19 BB dig. lox66DapAsubtilis lox66-DapAsubtilis P6 PCR product EcoRI PstI ~960 PCR product digested for cloning as a BioBrick Ø July 21
D19' BB dig. lox66DapAsubtilis lox66-DapAsubtilis P6' PCR product EcoRI PstI ~960 PCR product digested for cloning as a BioBrick 16 July 30
D20 bw-insert lox66-DapAE.coli lox66-DapAE.coli P5 PCR product XbaI PstI ~960 PCR product digested for usage as a backward insert Ø July 21
D21 bw-insert lox66-DapAsubtilis lox66-DapAsubtilis P6 PCR product XbaI PstI ~960 PCR product digested for usage as a backward insert Ø July 21
D21' bw-insert lox66-DapAsubtilis lox66-DapAsubtilis P6' PCR product XbaI PstI ~960 PCR product digested for cloning as a BI 34 July 30
D22 pSB1A2 open vector BBa_J61047 (MP9.1 & MP9.2) EcoI PstI Ø D22.1 Open pSB1A2 vector for BioBrick EcoRI/PstI insertio D22.1 Ø D22.2:59 July 23
D23 AraC/pBad promoter fw-insert BBa_I0500 (MP4.1 & MP4.2) EcoI SpeI 1233 AraC/pBad promoter ready for use as a forward insert Ø D23.1 93&53 July 23
D24 eCFP bw-insert BBa_E0422 (MP6.1 & MP6.2) XbaI PstI eCFP (RBS-OFR_LVA-T) ready for use as a backward insert 56&55 July 23
D26 gfp-tripart BBa_E0840 (MP8.1 & MP8.2) XbaI PstI gfp-tri part (strongRBS-ORF-T) ready for use as a backward insert 47&47 July 23
D27 lox71-ftsZ bw-insert lox71-ftsZ PCR product P7 XbaI PstI lox71-ftsZ(unmutated) ready for use as a backward insert Ø July 23
D27' lox71-ftsZ bw-insert lox71-ftsZ PCR product P7 XbaI PstI lox71-ftsZ(unmutated) ready for use as a backward insert 24 July 30
D28 RBS-DapAColi BB P8 (RBS-DapAColi) EcoRI PstI ~950 Biobrick 54 July 25
D29 RBS-DapAColi P8 (RBS-DapAColi) XbaI PstI ~950 Backward insert 44 July 25
D30 RBS-DapASubtilis BB P9 (RBS-DapASubtilis) EcoRI PstI ~950 Biobrick 72 July 25
D31 RBS-DapASubtilis P9 (RBS-DapASubtilis) XbaI PstI ~950 Backward insert 51 July 25
D32 Lox71-FtsA-FtsZ P10 Lox71-FtsA-FtsZ XbaI PstI ~2600 Backward insert - EcoRI site still presents : we can't use EcoRI 47 July 25
D33 RBS(B0030)CreORF L6' (clone 1 & 2) XbaI PstI RBS-Cre digestion for use as a BI 29&27 July 30
D34 lox66 L9' lox66 SpeI PstI Biobrick plasmid carrying lox66 digested as a BV 18 July 30
D35 lox71 L10' lox71 SpeI PstI Biobrick plasmid carrying lox71 digested as a BV July 30
D36 lox71-KmR-lox66 P11 lox71-KmR-lox66 EcoRI PstI lox71-KmR-lox66 digested as a biobrick insert July 30
D37 lox71-gfp(tripart) BI L12.1 & L12.2 miniprep products (from 30/07 ligation reaction) XbaI PstI lox71_RBS-gfp-double terminator extracted as a backward insert Aug. 6
D38 BB-insert lox66-DapAE.coli lox66-DapAE.coli in pTOPO (P5 PCR product)(clones 1+2) EcoRI PstI lox66-dapAE.coli extracted from pTOPO plasmid as a BB insert Aug. 6
D39' BI-insert lox66-DapAE.coli lox66-DapAE.coli in pTOPO (P5 PCR product)(clones 1+2) XbaI PstI lox66-dapAcoli extracted for usage as a backward insert Aug. 7
D40 BB-insert lox71-KmR-lox66 L25.1 miniprep product (from ligation rxn of 30/07 EcoRI PstI lox71-KmR-lox66 construct extracted for clonong as a biobrick Aug. 6
D41 araC/pBad-J61002 plasmid SP dig. L1.2, L1".1 & L1".2 miniprep product SpeI PstI J61002 plasmid containing araC/pBad promotor digested for insertion of a BI CDS Aug. 7

Unexpected digestion results

Unexpected results
Number Matrix Name Enzyme 1 Enzyme 2 Expected Size Description Date
D16 pSB1AK3::B0015 (MP3.1 & MP3.2) EcoR1 Pst1 ~3000 We have one band at ~2000. Furthermore, the strain which is suppose to be resistant to Kanamycin is not. We suspect that the part is in the pSB1A2 instead of the pSB1AK3 July 21
D17 pSB1A2::E0422 (MP6.1 & MP6.2) EcoRI PstI ~2040 + ~920 3 bands July 21
D25 BBa_E0421 (MP7.1 & MP7.2) PoPs to GFP converter XbaI PstI July 23

Strains (glycerol stock)

Strains glycerol stock
Number Name Description Verified clone Date
S1 MG1655 Standard E.Coli strain
S2 Acinetobacter ADP1 Strain used for TG coloration test with Red Nile
S3 DH5alpha pKs::DGAT Enzyme catalysing the transormation of DAG into TG
S4 BBa_pJ23100 Strong constitutive promoter in J61002 (clones 1 + 2)
S5 BBa_B0015 Double terminator (clones 1 + 2)
S6 BBa_pJ23107 Medium constitutive promoter in J61002 (clones 1 + 2) July 19
S7 BBa_B0030 Strong RBS (clones 1 + 2) July 19
S8 BBa_E0422 ECFP (RBS+LVA+Term) (clones 1 + 2) July 19
S9 BBa_E0241 PoPs to GFP converter (clones 1 + 2) July 19
S10 BBa_E0840 gfp tri-part; strong rbs (clones 1 + 2) July 19
S11 BBa_J61047 Cre ORF (clones 1 + 2) July 19
S12 W121 05/07/07
S13 BBa_I0500 pBAD AraC (clone 1+2) July 20
S14 BBa_pJ23107 Medium constitutive promoter in J61002 (clone 3+4) July 20
S15 BBa_P1003 Kanamycin resistance cassette (clone 1+2) July 20
S16 J61002::I0500 AraC pBad regulating mRFP expression in pJ61002 (L1) July 27
S17 pSB1A2::Lox66-DapA Coli Lox66-DapA Coli cloned as a biobrick (L2) July 27
S18 pSB1A2::B0030 + J61047 RBS(B0030)-Cre ORF (L6) (clones 1+2) July 27
S19 pSB1A2::BBa_J37026 Lox66 (L9)(1 clone) July 27
S20 pSB1A2::BBa_J37028 Lox71 (L10)(1 clone) July 27
S21 pSB1A2::BBa_J37028+BBa_E0840 Lox71-gfp tri-part (L12) L12.2 ok ?
S22 pSB1A2::Lox71-kmR-lox66 Lox71-kmR-lox66 (L25) ?
S23 pTopo::P5 Lox66-DapAColi ?
S24 pTopo::P6 Lox66-DapASubtilis ?
S25 pTopo::P8 RBS-DapAColi ?
S26 pTopo::P9 RBS-DapASubtilis ?
S27 J61002 August 14
S28 J61025 FRT-Cm-FRT August 14


  • p2C (CFP fluorescent)
  • p2Y (YFP fluorescent)
  • FtsZTS strains
    • MG1655 FtsZ -TS 84
    • 121.1
    • 121.2
    • 121.3
    • 121.4
    • 121.5
    • 121.6
    • 129
    • 1.129
    • DCR 14.1
    • DCR 14.2
    • DCR 14.3