Paris/Freezer
From 2007.igem.org
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 |
| 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 | |||
| 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 | |||
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) | ? | |
| 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
