Team:UT-Tokyo/Notebook
Contents
- 1 Protocol
- 1.1 Miniprep
- 1.2 Digestion
- 1.3 Gel Electrophoresis
- 1.4 Gel Purification
- 1.5 Ligation
- 1.6 Inoculation
- 1.7 Heat-Shock Transformation
- 1.8 Colony PCR
- 1.9 Chemically Competent Cell Preparation
- 1.10 LB Preparation
- 1.11 Agar Plate Preparation
- 1.12 Glycerol Stock Preparation
- 1.13 Sigma/ Anti-Sigma Assay
- 1.14 Pnrd Promoter Assay
- 2 Week 1
- 3 Week 2
- 4 Week 3
- 5 Week 4
- 6 Week 5
- 7 Week 6
- 8 Week 7
- 9 Week 8
- 10 Week 9
- 11 Week 10
- 12 Week 11
- 13 Week 12
- 14 Week 13
Protocol
Miniprep
Miniprep is done using the Wizard® Plus SV Minipreps DNA Purification System from Promega.
- Add 1.5 mL of overnight culture in a microcentrifuge tube
- Centrifuge at 15000 rmp for 1 min
- Discard suspension
- Add 1mL of overnight culture
- Centrifuge at 15000 rmp for 1 min
- Discard suspension
- Add 250 uL of Cell Resuspension Solution
- Vortex
- Add 250 uL of Cell Lysis Solution
- Invert
- Add 10 uL of Alkaline Protease
- Invert
- Leave for 3 min
- Add 350 uL of Neutralization solution
- Centrifuge at 15000 rmp for 10 min
- Place column in collection tube and transfer all suspension to the column
- Centrifuge at 15000 rmp for 1 min
- Discard flow-through
- Add 750 uL of Wash Solution
- Centrifuge at 15000 rmp for 1 min
- Add 250 uL of Wash Solution
- Set the column in a new microcentrifuge tube and discard the collection tube
- Add 100 uL of Nuclease-free water to the column
- Leave for 1 min
- Centrifuge at 13000 rmp for 1 min
Digestion
Add the following in a 1.5 mL microcentrifuge tube (from small to larger volume) and incubate at 37℃ for 2 hours.
| DNA | 500 ng |
| 10x Buffer | 2 uL |
| Restriction Enzyme | 0.5 uL each |
| MilliQ | up to 20 uL |
Gel Electrophoresis
- Prepare agarose gel
- Measure appropriate amount of agarose (1-2%) to 200 mL of TAE in a beaker
- Microwave until all agarose has dissolved
- Allow agarose solution to cool down
- Add 10 uL of ethidium bromide
- Pour solution into gel tray.
- Leave until gel has solidified
- Add corresponding amount of 6x loading dye (4uL to 20uL of digestion mixture) to each sample.
- Prepare ladder
- Promega 1 kb/100bp DNA Ladder 5 uL
- Loading Dye 1 uL
- Place agarose gel in electrophoresis machine and fill with 1x TAE until the gel is covered
- Load samples in the well
- Run gel at 110 V for 30 min-1 hour
Gel Purification
- Cut out desired band visualised under UV
- Place in 1.5 mL microcentrifuge tube
- Add 400 uL of Membrane Binding Solution
- Incubate at 60℃ for 10 min. Vortex every 2 min to melt gel.
- Set column in collection tube and apply solution into the column
- Leave for 1 min
- Centrifuge at 12000 rpm for 1 min
- Discard flow-through and add 750 uL of Membrane Wash Solution
- Centrifuge at 12000 rpm for 1 min
- Discard flow-through and add 500 uL of Membrane Wash Solution
- Centrifuge at 12000 rpm for 5 min
- Set column in new microcentrifuge tube
- Add 20 uL nuclease free water
- Leave for 1 min
- Centrifuge 15000 rpm for 1 min
Ligation
Add the following in a microcentrifuge tube and incubate at room temperature for 15min (for ° end ligation ), 5min (for cohesive end ligation).
| Vector | 100-200 ng |
| Insert | 2-3 times molarity of vector |
| T4 DNA Ligase | 1 uL |
| 2x Rapid Ligation Buffer | 5 uL |
| MilliQ | up to 10 uL |
Inoculation
The 1st ,2nd,and 3rd procedures are conducted in clean bench.
- Add 4 mL LB in a test tube.
- Add 4 uL AMP(50 ug/mL),1.3uL KAN(35 ug/mL),or CAM(33 mg/mL) into the LB.
- Pick colonies or glycerol stock and pipette in the LB.
- Incubate at 37℃ overnight.
Heat-Shock Transformation
- Add 10 uL of DNA/ligation product into 100 uL of competent cell
- Leave on ice for 30 min
- Heatshock at 42℃ for 45 seconds
- Leave on ice for 2 min
- Add 1 mL of LB (skip this step for AMP)
- Incubate at 37℃ for 30 min
- Centrifuge for at 15000 rpm for 1 min
- Remove 800 uL of suspention
- Spread on plate
- Incubate at 37℃ overnight
Colony PCR
1.Prepare the following master mix
| Milli Q | 3 uL/sample |
| 2x GoTaq Mix | 5 uL/sample |
| 10x VR | 1 uL/sample |
| 10x VF2 | 1 uL/sample |
| Total | 10uL/sample (*prepare mixture for 1 sample extra) |
2. Aliquot 10uL each to PCR tubes
3. Pick colonies and pipette in mixture
4. Set up the PCR machine
| 95℃ | 3 min |
| *95℃ | 30 sec |
| *52℃ | 30 sec |
| *72℃ | 1 min/1kbp |
| *72℃ | 5 min |
| *repeat | 30 cycle |
| 4℃ | forever |
5. Run samples by electrophoresis
Chemically Competent Cell Preparation
- Preculture cells in 2 mL LB overnight
- Add 500 uL of the overnight culture in 50 mL of fresh LB in a conical flask x2
- Incubate at 37℃ for around 3hours (until OD is 0.5).
- Cool the culture on ice for 30 min
- Transfer culture to a 50 mL centrifuge tube and centrifuge at 3200 rpm for 5 min
- Discard the suspension
- Add 25 mL of 50mM CaCl2 to the pellet and resuspend
- Cool the culture on ice for 1 hour
- Centrifuge at 3200 rpm for 5 min
- Discard suspension
- Add 5 mL of 50 mM CaCl2(cooled) and resuspend
- Aliquot 100 uL each to a microcentrifuge tube
- Store at -80℃
LB Preparation
Add the following in a reagent bottle and autoclave the bottle
| LB-Bouillon,Miller | 12.5 g |
| Milli Q | 500 mL |
Agar Plate Preparation
- Add the 200 mL Milli Q,5g LB-Bouillon, 3 g Agarose and a magnetic rotor into 300ml beaker and autoclave it.
- Agitate the solution by magnetic stirrer and cool it
- Before the solution are solidified, add 200 uL AMP(50 ug/mL), 200 uL KAN(35 ug/ml),or 65 uL CAM (100 ug/mL) into the solution, and divide the solution into 10 plates in the clean bench
- Cool it until these agar plates are solidified completely
Glycerol Stock Preparation
- Add 300 uL 50% glycerol(glycerol:Milli Q=1:1(vol.)) and 700 uL culture medium into a sterile microcentrifuge tube with a screw cap
- Store at -80 ℃
Sigma/ Anti-Sigma Assay
- Incubate cells overnight in 3mL of liquid LB at 37 °C
- Add 100 uL of the overnight culture in 3 mL of fresh M9 with appropriate antibiotics the next day
- Incubate at 37 °C
- Dilute appropriately so that fluorescence level falls within the detectable range of the spectrofluorophotometer
- Measure OD600 and fluorescence (excitation 488 nm; emission 516 nm) when the OD reaches about 0.4 (approximately 4 hours after dilution)
Measurements were done using Spectrofluorophotometer RF-5301PC from Shimazu Scientific Instruments.
Pnrd Promoter Assay
- Incubate cells overnight in 3 mL of liquid LB at 37 °C
- Add 100 uL of the overnight culture in 3 mL of fresh M9 with appropriate antibiotics the next day
- Incubate at 37 °C
- Dilute 81 fold before measurement.
- Measure OD600 and fluorescence (excitation 488 nm; emission 516 nm) every 9 minutes for 1.5 hour when the OD reaches about 0.4 (approximately 4 hours after dilution)
Measurements were done using Spectrofluorophotometer RF-5301PC from Shimazu Scientific Instruments.
Week 1
Day 1- 07/11/16
Made 5mL of 1000x ampicillin stock(50mg/mL) solution, plates
Made LB
Day 2- 07/12/16
Made competent cells
Made 5mL 1000x chloramphenicol stock(100mg/mL), plates
Transformation of bacteria with plasmid from kitplate
| Pconst | Bra_J61002 |
| RBS | pSB1C3 |
| dTerm | pSB3K3 |
| GFP-LVA | pSB1C3 |
| RBS-GFP-dTerm | pSB1C3 |
Day 3- 07/13/16
Transformations made on the previous day had failed.
Remade chloramphenicol plates after realizing that the concentration was wrong. The stock solution made was 3000x not 1000x.
Redid the transformations attempted on the previous day.
Overnight culture of cells for competent cells batch 2
Day 4- 07/14/16
Made competent cells.
Inoculation of cells transformed on the previous day
Day 5- 07/15/16
Miniprep of inoculated cells.
Made glycerol stock of inoculated cells.
Digestion
| Pconst | S+P |
| dTerm | E+X |
| GFP-LVA | E+S |
| RBS-GFP-dTerm | X+P |
Week 2
Day 1- 07/18/16
Holiday
Day 2- 07/19/16
Gel purification of gel electrophoresis product (from 07/15/16).
Did a nanodrop of the purified product. The concentration was quite low so the digestion was done again, the bands could not be seen clearly but the gel was cut where the bands were supposed to be there. Some concentration of DNA could be measured after purification.
Ligation Pconst + RBS-GFP-dTerm, GFP-LVA+dterm
Transformation of ligated product and pSB3K3-I20260 (from kitplate 4 18A)
Day 3- 07/20/16
Transformation done on the previous day on the CAM plate had all failed.
AMP plate was covered in cell colonies. It seemed like it was contaminated. Transformation of pSB3K3-I20260 was successful.
Ligation was re-attempted using the digested products from yesterday.
Transformation was done again, this time using competent cells batch 2.
Inoculation of pSB3K3-I20260 and pconst
Day 4- 07/21/16
Transformation of Pconst + RBS-GFP-dTerm had failed (no colonies on plate), GFP-LVA+dterm(cu with seemed to be successful.
Digestion of Pconst + RBS-GFP-dTerm was reattemped.
Inoculation of gfplva-dterm
Day 5- 07/22/16
Gel electrophoresis of product digested on the previous day. For RBS-GFP-dterm, only one band was seen, probably the wrong enzyme was used or the enzyme is damaged.
Made glycerol stock of gfplva-dterm.
Ligated and transformed pconst-RBS-GFP-dterm
Week 3
Day 1- 07/25/16
No colonies for pconst-RBS-GFP-dterm
Digestion of rbs and GFPlva
Made kanamycin stock 5mg/mL
Colony pcr of gfp-lva dterm-> unsuccessful it appeared to be gfp-lva
Day 2- 07/26/16
Ligation of rbs- GFPlva and pconst
Made more CAM plates
Inoculation of pconst-rbs-gfp-dterm, pSB3K3-I20260
Inoculation of cell culture D2, D3, E17, E18 from 2014
Day 3- 07/27/16
PCR of D2, D3, E17, E18-> E17 showed unexpected bands, others were as expected
Miniprep of D2, D3, E17, E18, pSB3K3-I20260
Made new glycerol stock for cell cultures with the above constructs and one for I20260-pSB3K3
Digestion of D2, D3, pconst.
Ligation of pconst-D2, pcont-D3
Transformation of yesterday’s ligation (RBS-GFPlva) and pconst weak(from kit plate4 4-E)
Inoculation of pconst, E17, E18, E6, dterm, RBS
Found out that the LB was contaminated, transformations and inoculations done previously may have been affected.
Day 4- 07/28/16
Transformation of pconst-D2, pconst-D3 were successful
Miniprep of pconst, E17, E18, dterm, RBS . Nothing had grown in the overnight culture of E6.
Made glycerol stock of E17, E18 again as previous one might have been made in contaminated LB
Digestion of GFPlva-pSB1C3 and Pconst-rbs-gfp-dterm (X+P)
Ligation of the above digested parts.
Proper bands did not appear in the gel electrophoresis image of Pconst rbs gfp dterm (it was not cut at all)
It was found out later that the sequencing results of the cut sites of the plasmids were not correct
Transformation of the ligated product
Inoculation of Pconst-D2, Pconst-D3
Week 4
Day 1- 08/01/16
Last week’s transformations were all successful.
Colony PCR rbs-GFPLVA,PconstWeak,E17,E18-> not quite sure for pconst weak, rbs-GFPlva. E17, E18 seem to be ok
Digestion of J23101-D2, J23101-D3, GFPlva-SB1C3 (E+S)
Ligation pSB1C3+PconstD2,pSB1C3+PconstD3
Transformation of the ligated product
Inoculation of RFPlva, PconstWeak, RBS-GFPlva, Pconst-RBS-GFP-dterm,A9,A10
Day 2- 08/02/16
Yesterday’s transformation had all failed
Miniprep (A9,RBS-GFPPlva,RFPlva,Pconst-RBS-GFP-dterm,PconstWeak)
Made glycerol stock
Digestion (rbs-GFPlva, pconst, pconst weak, d2, d3)
PCR（GFPlva,RBS-GFPlva,RBS-GFPlva2,pconstweak）-> Pconstweak seems ok, no bands for GFPlva, incorrect band for gfplva
Messed up gel extraction
Inoculation of A10(amp),PRGD(KAN),D2,D3,pconstweak(CAN)
Day 3- 08/03/16
Miniprep (A10(amp),PRGD(KAN),D2,D3,pconstweak(CAN)
Digestion(Pconst-BBa_J61002,Pconstweak,D2,D3,pconst-D2,Pconst-D3,dterm,GFPlva,RBS-GFPlva,PRGD-pSB3K3). Concentration of the purified product was low, possibly because he wrong buffer was used.
Ligation(PconstD2+3K3,PconstD3+3K3,GFPlva+61002,RBS-GFPlva+dterm,Pconstweak+D2,Pconstweak+D3)
Transformation of ligated product
Inoculation (Pconstweak,PRGD)
Day 4- 08/04/16
Only transformation for RBS-GFPlva-dterm and GFPlva-J61002 was successful
Miniprep(PRGd-3K3,Pconstweak)
PCR (GFPlva-BBa_J61002×2,GFPlva×2,RBS-GFPlva-dterm×2). Rbs-GFPlva-dterm turned out to be just dterm. For other colonies, no bands were shown, extention of the extension time maybe needed.
Digestion (Pconstweak,D2,D3,PRGd-3K3)S+P
The results for the gel electrophoresis of the digested product was not quite right so ligation was not done.
Inoculation (PconstWeak,RBS-GFPlva-dterm×3,PconstD2,PconstD3,GFPlva-BBa_J61002×2)
Day 5- 08/05/16
Miniprep (PconstWeak,RBS-GFPlva-dterm×3,PconstD2,PconstD3,GFPlva-BBa_J61002×2).
Digestion (PRGd-3K3,PconstD2,PconstD3,PconstWeak,D2,D3,dterm,GFPlva-61002)
Ligation (PconstWeak-D2,PconstWeak-D3)
Transformation of ligated product
Week 5
Day 1- 08/08/16
PCR of PconstWeak-D2,PconstWeak-D3. Bands for Pconst weak appeared for some of the colonies, while nothing appeared for most others.
Digestion ( PRGd-3K3, pconstD, GFPlva-J61002, rbs)
Made KAN + CAM plates
Gel electrophorsis of digested product. 3k3 was not cut at all, GFPlva was not cut properly, cant see PconstD2. Ecor1 Spe1may be damaged
Transformation RBS-GFPlva
Inoculation PRGd10ml,GFPlva-61002-4,PconstweakD2,PconstweakD3,E17,E18
Preculture for competent cells
Day 2- 08/09/16
Glycerol stock (GFPlva-61002-4,PconstweakD2,PconstweakD3)
Miniprep(PRGd,GFPlva-61002-4,PconstweakD2,PconstweakD3,E17,E18)
Digestion(E17,E18,PconstweakD2,PconstweakD3,PconstD2×2,PconstD3×2,3K3)(E17,E18:vector). E18 is alright, E17 is too light, no correct bands for Pconst D2 PCR(RBS-GFPlva×3,PconstWeakD2×2,PconstWeakD3×2). All rbsgfplva turned out to be rbs. PconstweakD2,PconstweakD3 seems to be correct.
Made competent cells
Ligation and transformation(PconstweakD2E17, PconstweakD3E18, PconstD2E17, PconstD3E18, PconstD2E18,PconstD3E17,E17-3K3,E18-3K3,CFPlva(kitplate4-5D))
Attempted double transformation(PRGd-3K3,E17-pSB1C3)
Day 3- 08/10/16
Miniprep(RBS-GFPlva×3)
Day 4- 08/11/16
Public Holiday
Day 5- 08/12/16
Public Holiday
Week 6
Day 1- 08/15/16
Public Holiday
Day 2- 08/16/16
Public Holiday
Day 3- 08/17/16
PCR (PconstweakD2E17, PconstweakD3E18, PconstD2E17, PconstD3E18, PconstD2E18,PconstD3E17,E17-3K3,E18-3K3). -> most did not seem to contain the right construct. Except rbs-gfplva. Perhaps the Spe1 has gone bad.
Made CAM plates
Check of restriction enzymes. All the enzymes seemed to work fine.
Inoculation (E18&PRGD(CAM)(KAN)(CAM+KAN), CFPlva(AMP), RBS-GFP-dterm(CAM))
Day 4- 08/18/16
All cultures grew for the inoculation. Double transformation seemed be successful.
Miniprep (E18+PRGd,CFPlva)
Digestion (RBS-GFPlva, RBS-GFP-dterm, dterm, E18+PRGd, CFPlva, Pconst, Pnrd). All except Pconst had undigested fragments. The one with double transformation also showed proper bands
PCR (PconstD2-3K3, PRGd-3K3.) Showed mysterious band of slightly less than 500bp.
Ligation(RBS-GFPlva-dterm, RBS-GFPlva-pSB1A2, Pnrd-61002, Pconst-RBS-GFP-dterm, Pnrd-1A2, RBS-GFPlva)
Transformation (RBS-GFPlva-dterm, RBS-GFPlva-pSB1A2, Pnrd-61002, Pconst-RBS-GFP-dterm, Pnrd-1A2, RBS-GFPlva)
Inoculation (dterm, Pconst, PconstD2, RBS)
Day 5- 08/19/16
PCR (PconstD2-3k3,PRGd-3K3(plasmid),Pconst-RBS-GFP-dterm-1C3, Pconst-RBS-GFPlva). Pconst-RBS-GFPlva seems OK. PconstD2-3k3 is possibly PRGd-3k3. Pconst-RBS-GFP-dterm is OK)
Miniprep (Pconst,pconstD2,RBS,dterm)
Digestion(E17,E18,PconstD2,PconstD3,RBS-GFPlva, dterm, Pconst-61002,PRGd-3K3,CFPlva-1A2). Digestion results are still not too good, some plasmid remains uncut.
Transformation E17PconstD2, E18PconstD2, E17PconstD3, E18PconstD3, Pnrd-1C3, RBSGFPlvadterm(CAM), PconstD2-3K3, PconstD3-3K3(KAN), Pnrd-61002, RBSGFPlva-1A2(AMP)
Week 7
Day 1- 08/22/16
PCR (E17PconstD2, E18PconstD2, E17PconstD3, E18PconstD3, Pnrd-1C3, RBSGFPlvadterm(CAM), PconstD2-3K3, PconstD3-3K3(KAN), Pnrd-61002, RBSGFPlva-1A2(AMP))
Made 3% agarose gel
Made AMP plates
Transformation(PconstD2-3K3, E17-PconstD2, E17PconstD3, RBSGFPlvadterm)
Preculture(160818:Pconst-RBS-GFP-dterm(AMP), RBS-GFPlva-dterm(CAM) 160819:E18-PconstD2(光る),Pnrd1C3×2, E17-PconstD3, E18-PconstD2(CAM), PconstD3-3K3(光らない)(KAN), Pnrd-61002, RBS-GFPlva-dterm(AMP))
Preculture for competent cells
Day 2- 08/23/16
Only PconstD2-3K3, RBS-GFPlva-dterm grew colonies for yesterday’s transformation.
MIniprep (160818:Pconst-RBS-GFP-dterm(AMP), RBS-GFPlva-dterm(CAM) 160819:E18-PconstD2(光る),Pnrd1C3×2, E17-PconstD3, E18-PconstD2(CAM), PconstD3-3K3(KAN), Pnrd-61002, RBS-GFPlva-dterm(AMP))
Digestion(E17.E18,PconstD2,PconstD3,PRGd-3K3). Pconst D2 was not cut. No bands apeared for ToeholdGFPlva and PconstTrigger Made competent cells
Ligation E17+PconstD3, 1A2+Pnrd, RBS-GFPlva+dterm
Transformation (E17PconstD3, RBS-GFPlva-dterm(CAM), Pnrd-1A2, PconstTrigger1, ToeholdGFPlva(AMP))
Double transformation(PcosntD3-3K3+E18(KAN+CAM))
Preculture(PconstD2, PRGd-3K3, E17, E18)
Day 3- 08/23/16
Miniprep(PconstD2, E17, E18, PRGd-3K3)
PCR(PconstD2-3K3(not OK; unknown band seen at around 400bp), Pnrd-1A2(ok), RBS-GFPlva-dterm(ok), E18+PconstD3(not ok))
Digestion(PcosntD2, PcosntD3, PconstWealD2, PcosntWeakD3, E17, E18, PRGd3K3)
Ligation(PconstD2-E17, PconstD2-E18, PconstD3-E17, RBSGFPlva-dterm)
E17-PconstD maybe slightly contaminated with E18.
Transformation(E17-PconstD2, E18-PconstD2, E17-PconstD3, RBSGFPlva-dterm)(CAM)
Preculture(PconstTrigger1, toeholdGFPlva, Pnrd-1A2(AMP), PconstD2-3K3(KAN), PconstD3-3K3+E18(CAM+KAN)PcWD2, PcWD3(CAM))
Day 4- 08/24/16
Glycerol stock (PconstTrigger1, toeholdGFPlva, Pnrd-1A2, PconstD2-3K3 PconstD3-3K3+E18)
Miniprep(PconstTrigger1, toeholdGFPlva, Pnrd-1A2, PcWD2, PcWD3, PconstD2-3K3)
PCR(E17-PconstD3(not OK), PconstD2-3k3(not OK), PconstD3-3K3, PRGd-3K3, RBS-GFPlva-dterm(not OK))
Digestion(toeholdGFPlva,Pnrd1A2, RBSGFPdterm)(E17,E18,PconstD2,PconstD3,PcWD2,PcWD3)
Ligation(toeholdGFPlva-dterm, RBS-GFPlva-dterm, Pnrd-1C3, E17PcWD2, E18PcWD3, PcWD2-3K3, PcWD3-3K3, PconstD2-3K3)
Made AMP plates
Transformation(toeholdGFPlva-dterm, RBS-GFPlva-dterm, Pnrd-1C3, E17PcWD2, E18PcWD3(CAM), PcWD2-3K3, PcWD3-3K3, PconstD2-3K3(KAN), PconstD23K3+E17(CAM+KAN))
Preculture(E17PconstD3×2 )
Day 5- 08/25/16
Glycerol Stock (E17PconstD3-6)
Miniprep(E17PconstD3-6,-7)
PCR(PcWD3-3K3(unknown), PcWD2-3k3(unknown), E18PcWD3(not OK), E17PcWD2(not OK), E17PconstD2(not OK), E17PconstD3(not OK))
Digestion(dterm-3K3, PconstRBSGFPdterm-1A2)
Ligation(RBSGFPlva-dterm, toeholdGFPlva-dterm, Pnrd-1A2, PconstRBSGFPdterm-1C3)
Transformation(RBSGFPlva-dterm, toeholdGFPlva-dterm, PconstRBSGFPdterm-1C3(CAM), Pnrd-1A2(AMP), PconstD2-3K3+E17(KAN+CAM))
Week 8
Day 1- 08/29/16
PCR(RBS-GFPlva-dterm(OK), toeholdRBSGFPlva-dterm(not OK), Pnrd-1A2(not OK), PconstWD3-3K3(not OK), PconstWD2-3K3(not OK), E18-PconstD2(not OK))
Digestion(PconstRBSGFPdterm1A2, Pnrd, sigma16, sigma35, antisigma16, antisigma33, dterm, E18-1C3, RBSGFPlva-1A2)
Ligation (Pnrd-1C3)
Transformation(Pnrd-1C3, dterm)
Preculture(D8, D9) (E18PcWD3, E17PcWD2, PcWD2-3K3, PcWD3-3K3, RBSGFPlva-dterm)
Day 2- 08/30/16
Glycerol Stock (E18PcWD3, E17PcWD2, RBSGFPlva-dterm, D8, D9)
Miniprep(E18PcWD3, E17PcWD2, RBSGFPlva-dterm, D8, D9)
PCR(PcWD3-3K3, PcWD23K3, toeholdGFPlvadterm(NG), Pnrd1C3(OK), PRGd-3K3(OK))
Digestion(PconstRBSGFPdterm, RBSGFPlva-dterm, D8, D9, Pconst)
Ligation(Pconst-D8, Pconst-D9, Pconst-RBS-GFPlva-dterm, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Pnrd-1A2, PconstRBSGFPlva-1C3)
Transformation (Pconst-D8, Pconst-D9, Pconst-RBS-GFPlva-dterm, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Pnrd-1C3, PconstRBSGFPlva-1C3)
Preculture (Pnrd-1C3, PconstRBSGFPdterm, PconstD3, PcWD2-3K3, PcWD3-3K3、PcD2-3K3)
Day 3- 08/31/16
Glycerol Stock (Pnrd-1C3, PcD2-3K3)
PCR(Pconst-D8, Pconst-D9, Pconst-RBS-GFPlva-dterm, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Pnrd-1C3(OK), PconstRBSGFPdterm-1C3, PconstD2-3K3 )
Miniprep(Pnrd-1C3, PconstRBSGFPdterm, PconstD3, PcD2-3K3)
Digestion(PconstRBSGFPdterm, Pnrd-1C3, RBS, PRGd-3K3, RBSGFPlvadterm, RBSGFPdterm, PcWD2-3K3, PcWD3-3k3)
Transformation (PcWD2-3k3, PcWD3-3k3, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2)
Preculture(PconstD8, PconstD9, PconstRBSGFPlvadterm, sigma35-1A2, Pnrd-1C3, PconstRBSGFPdterm-1C3)
Double transformation(E17-PcD2)
Day 4- 09/01/16
LB was contaminated. Made new batch of LB.
Double transformation failed
PCR(Pnrd-1C3(OK), PconstRBSGFPdterm1C3, sigma35(NG))
Digestion(PconstRBSGFPdterm1A2, sigma16, sigma35, antisigma16, antisigma33, Psigma16, Psigma30, E17, PconstD2)
Transformation(sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Psigma16-1A2, Psigma30-1A2, E17PconstD2)
Preculture(PconstRBSGFPlvadterm, PconstD8, E17, JM109, PcWD2)
Day 5- 09/02/16
Glycerol Stock (Pnrd-1C3, PconstRBSGFPlvadterm)
PCR (sigma 16(OK), antisigma33(OK), Psigma16(unknown), PcWD2-3k3(unknown))
Digestion(Pnrd-1C3, PconstD8, PconstD9, PconstD2, E17, E18, sigma35-1A2(0901)
Ligation(Pnrd-RBSGFPdterm, Pnrd-RBSGFPlvadterm, PconstD8E17, PconstD9E18, PconstD2E17, Psigma30-1A2, sigma35-1A2)
Transformation(Pnrd-RBSGFPdterm, Pnrd-RBSGFPlvadterm, PconstD8E17, PconstD9E18, PconstD2E17, Psigma30-1A2, sigma35-1A2)
Week 9
Day 1- 09/05/16
PCR sigma35-1A2(NG), E18PconstD9(OK), E17PconstD8(OK)
Digestion(Pnrd-1A2, PconstD2, D2, Psigma30, Pconst, PcW)
Transformation(Pconst+D2, Pnrd+RBSGFPlvadterm, PcW+RBSGFPdterm, PcW+RBSGFPlvadterm, Psigma30+1A2)
Preculture (PcWD2-3K3, PcWD3-3K3, sigma16-1A2, sigma35-1A2, Psigma16-1A2, antisigma16-1A2, antisigma33-1A2, E17PconstD8, E18PconstD9, JM109)
Day 2- 09/06/16
Glycerol Stock (sigma16-1A2, sigma35-1A2, Psigma16-1A2, antisigma16-1A2, antisigma33-1A2, E17PconstD8, E18PconstD9)
PCR(E178(OK), E17PconstD8(OK), E18(OK), E18PconstD9(OK))
Miniprep(sigma16-1A2, sigma35-1A2, Psigma16-1A2, antisigma16-1A2, antisigma33-1A2, E17PconstD8, E18PconstD9)
Digestion(Pnrd, RBSGFPdterm, RBSGFPlvadterm, Pconsttrigger1dterm, toeholdGFPlva, PconstRBSGFPdterm, dterm, sigma16, sigma35, antisigma16, antisigma33, Psigma16, RBS)
ligation(PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3, toeholdGFPlva-dterm, RBS-sigma16, RBS-anti16, RBS-anti33)
Transformation(PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3, toeholdGFPlva-dterm, RBS-sigma16, RBS-anti16, RBS-anti33)
Day 3- 09/07/16
Holiday
Day 4- 09/08/16
PCR (PcW-RBSGFPdterm(OK), Pconsttrigger1dterm-1C3(NG), toeholdGFPlva-dterm(NG), RBS-sigma16(NG), RBS-anti16, RBS-anti33(OK))
Digestion(sigma351A2, sigma35,Psigma30, Pnrd, RBSGFPlvadterm, RBSGFPdterm, PconstRBSGFPdterm、RBS)
Ligation (pnrd-rbsgfpdterm, Pnrd-rbsgfplvadterm, rbs sigma 35, psigma30-1A2)
Transformation of ligated product
Preculture(PconstD2, PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3, toeholdGFPlva-dterm, RBS-sigma16, RBS-anti16, RBS-anti33)
Day 5- 09/09/16
Glycerol stock (PconstD2, PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3
Miniprep (PconstD2, PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3)
Digestion (pconst, rbs anti16, rbs anti33, rbs sigma16, toeholdgfplva)
Ligation (Pconstrbs-anti33)
Transformation PconstRBS-anti33
Week 10
Day 1-09/12/16(Nasuda,Sakata,Ishida,Chin)
Digestion (Pconsttrigger1dterm, 1C3, toeholdgfplva, dterm, RBS, sigma16, RBS, anti16 )
PCR (pc-rbs-anti-33(ok), rbs sigma 35(ok))
Gel extraction (Pconsttrigger1dterm, toeholdgfplva, dterm, RBS, sigma16, anti16, 1C3)
Ligation (Pconsttrigger1dterm-1C3, RBS-sigma16, RBS-anti16)
Preculture (pnrd-gfp, pnrd-gfplva , Psigma30-1A2, rbs-sigma35, Pcr pc-rbs-anti-33) Transformation (Pconsttrigger1dterm1C3, RBS-sigma16, RBS-anti16, PconstD2)
Day 2-09/13/16(Nasuda,Sakata,Hamakawa)
All cultures grew.
Miniprep (PnrdRBSgfpdterm, PnrdRBSgfplvadterm, Psigma30-1A2, RBS-sigma35)
Glycerol Stock (PnrdRBSgfpdterm, PnrdRBSgfplvadterm, Psigma30-1A2, RBS-sigma35)
PCR (Pconsttrigger1dterm, RBS-sigma16, RBS-anti16, PconstD2)
Digestion (Pconst, PconstWeak, RBSsigma35, Psigma30, RBSgfpdterm, RBSgfplvadterm, Pnrdrbsgfplvadterm, PnrdRBSgfpdterm, PconstD33K3, PconstRBSgfplvadterm)
Gel extraction (Pconst, PconstWeak, RBSsigma35, RBSgfplvadterm, Pnrdrbsgfplvadterm, PnrdRBSgfpdterm, PconstRBSgfplvadterm)
Preculture (PconstD2, anti35-1A2, toeholdgfplva, Pconsttrigger1dterm, RBS-sigma35, RBSgfpdterm, RBSgfplvadterm, PconstD2-3K3, PconstD3-3K3)
Ligation transformation (RBS-sigma16, RBS-anti16)
Day 3-09/14/16(Sakata,Nasuda,Ishida,Satou)
RBS-anti16 grew.
Miniprep (RBS-sigma35, anti35-1A2, Pconsttrigger1dterm-1C3, PconstD2,RBSgfpdterm,
toeholdgfplva, RBSgfplvadterm, PconstD3-3K3(PconstD2-3K3 failed))
Preculture (PconstD2-3K3)
Glycerol Stock (RBS-sigma35, anti35-1A2, Pconsttrigger1dterm-1C3, PconstD2)
PCR (RBS-anti35(NG), PconstD2-3K3(NG), PconstD3-3K3, PconstD2)
Digestion (PconstD3-3K3, anti35, PconstD2-1A2, toeholdgfplva, RBSsigma35,Psigma30, RBSgfpdterm, RBS)
Gel extraction (3K3, anti35, PconstD2, toeholdgfplva, RBSsigma35, Psigma30, RBSgfpdterm, RBS)
Day 4-09/15/16(Hamakawa)
PCR (RBSsigma35,RBSanti35,PconstD2-3K3,PnrdRBSgfplvadterm3K3(ok),
PnrdRBSgfpdterm3K3(ok),RBSsigma16,RBSanti16,Pconst-toeholdgfplva)
Preculture (PcWRBSgfpdterm,PconstRBSgfpdterm1C3,PconstRBSgfpdterm3K3,
PnrdRBSgfpdterm1C3,PnrdRBSgfpdterm3K3,PnrdRBSgfplvadterm1C3,PnrdRBSgfplvadterm3K3)
Day 5-09/16/16(Hamakawa)
Glycerol Stock(PnrdRBSgfpdterm3K3,PnrdRBSgfplvadterm3K3)
Fluorescence measurement in Ikeuchi lab.
(PcWRBSgfpdterm,PconstRBSgfpdterm1C3,PconstRBSgfpdterm3K3, PnrdRBSgfpdterm1C3,PnrdRBSgfpdterm3K3,PnrdRBSgfplvadterm1C3,PnrdRBSgfplvadterm3K3)
Week 11
Day 1-09/19/16
Holiday
Day 2-09/20/16(Nasuda,Hamakawa)
Digestion (anti35,PconD3-3K3,Pconst-RBSgfpdterm1A2,PconstWeakRBSgfpdterm1c3, PconstWeak)
PCR (RBS-sigma16,RBS-anti35,RBS-anti16,RBS-sigma35)
Gel extraction
Ligation (RBS-sigma16,RBS-anti16,RBS-anti35,1A2-anti35,PconstWeakgfplvadterm) Transformation (RBS)
Preculture
Day 3-09/21/16(Sakata,Hamakawa)
Miniprep (RBS-1c3, Pconsttoeholdgfplva-1A2, Pconst D2-3k3)
Digestion (Pconst-1A2, RBS-1c3, Pconst trigger, Pconsttoeholdgfplva-1A2, Pconst D2-3k3, E17, sigma35, sigma16, dterm)
Gel extraction
Ligation (PconstRBS-1A2, Pconsttriggger, PconsttoeholdgfpLva-1A2, PconstD2-E17, sigma16dterm)
Transformation (PconstRBS-1A2, Pconsttriggger, PconsttoeholdgfpLva-1A2, PconstD2-E17, sigma16dterm,anti16,anti35,sigma16)
Day 4-09/22/16
Holiday
Day 5-09/23/16(Sakata)
All cultures other than sigmadterm grew.
PCR (E17+Pconst D2(NG), Pconst trigger+Pconst THgfp(NG), anti 33(ok), PconstRBS, rbs sigma16, rbs- anti16(NG), rbs anti 33(NG), anti16(ok), sigma 16(ok) Ligation (Sigma16dterm, dterm, pcosnttriggerdterm, E17-PconstD2, E17)
Transformation (Sigma16dterm, dterm, pcosnttriggerdterm, E17-PconstD2, E17)
Week 12
Day 1-09/26/16(Sakata)
PCR (Pconstrbs(ok), Pconst triggerPconst toehold(NG), E17PconstD2(NG))
Preculture (Pconstrbs, antis33, anti16, sigma16, dterm, PconstriggerPconstTHgfp, RBS, A10, Pconst)
Day 2-09/27/16(Sakata,Nasuda)
Miniprep (dterm 1C3, RBS 1C3, Pconst RBS, "Pconst trigger Pconst RB", anti16, anti35, A10(Psigma11)）
Digestion (Psigma 16, anti35,sigma16,anti16,Pconst rbs, Psigma11, rbsgfpdterm, Pconst triggerPconstTHgfp, PconstD3-3K3)
Gel Extraction File:T--UT-Tokyo-Labnote-0927 PCR (Psigma 16, anti35,sigma16,anti16,Pconst rbs, Psigma11, rbsgfpdterm(NG), Pconst triggerPconstTHgfp(NG), PconstD3-3K3(NG))
Ligation (Pc-anti35, Pc-sigma16, Pc-anti16, P16-1A2, P11-RGdT)
Day 3-09/28/16(Sakata,Nasuda,Narita)
Miniprep (rbs gfp dterm, Pconst trigger, Pconst THgfp)
Digestion (Pconst triggerdterm , Pconst Toehold gfp )
Transformation (double Pconst triggerdterm 1C3 +Pconst Toehold gfp 1A2)
PCR (Psigma11-RBSgfpdterm(ok),PconstRBS-anti16(NG),PconstRBS-sigma16(ok), Psigma16-1A2(ok),PconstRBS-anti35(ok),Pconst trigger(ok))
Preculture (Psigma11-RBSgfpdterm,PconstRBS-anti16,PconstRBS-sigma16, Psigma16-1A2,PconstRBS-anti35,Pconst trigger)
Day 4-09/29/16(Sakata)
Miniprep (Pc-anti35, Pc-sigma16, Pc-anti16, P16-1A2, P11-RGdT)
Digestion (Pbad trigger dterm,dterm , Pnrdgfpdt-1c3,Pc-anti35 ,Pc-sigma16, Pc-anti16, P11rgdt X+P, PcRBSgfpdterm-3k3 X+P , Pc trigger , Pc TH gfp)
Gel Extraction File:T--UT-Tokyo-Labnote--0929 Ligation (P16-1c3, Pc anti16 dT, Pc anti35 dT, Pc sigma 33 dT, P11rgdT-3k3, Pbad trigger dterm -1C3)
Transformation (P16-1c3, Pc anti16 dT, Pc anti35 dT, Pc sigma 33 dT, P11rgdT-3k3, Pbad trigger dterm -1C3)
Day 5-09/30/16(Sakata)
All cultures other than P11rgdT-3k3 grew well.
PCR (Pc trigger dt, Pc TH gfp, Pnrd rgdt 1C3, Pc rgdt 3k3)
Week 13
Day 1-10/03/16(Sakata,Narita,Sanada,Nasuda)
Digestion (Pnrd gfp 1C3, Pcsigma16, Pc anti16, Psigma11 rgdt, Prgdt 3k3, Pc trigg,
Pc anti35, PcWrgdt 1C3, Pconst TH gfp)
Gel Extraction File:T--UT-Tokyo-Labnote--1003
Ligation (Pc sigma 16-dterm, Psigma11rgdt-3k3, PbadtriggsT-1C3, Pc anti35-dt, Pc anti16-dT, Pconst trigger-Pconst TH gfp) Preculture (PcRBSanti16dterm, pcRBSanti35dterm, Psigma16, RBSgfpdterm, Pbad) Transformation (1C3,1C3,1C3,3K3,Pconsttrigger1dtermPconsttoeholdgfplva-1A2)
Day 2-10/04/16(Sakata,Ikeda,Nasuda,Sanada)
Miniprep
Glycerol Stock (Pc rbs anti16 dt, rbs gfpdt, Pbad, Pc rbs anti35dt, Psigma16)
Digestion (Pnrd gfp 1c3, rbs sigma16, rbs sigma 35, Pconst rbs sigma 16dT, Pconst rbs sigma 35 dT, Psigma16 rbs gfp dT, Psigma35 rbs gfp dT, P16, TH gfp, Pbad trigger, Pbad, Pc trigger, Pc THgfp)
Gel Extraction FileT--UT-Tokyo-Labnote--10041 File:T--UT-Tokyo-Labnote--10042
Ligation (rbs16-1C3, rbs35-1c3, Pc rbs sigma16dt-iC3, Pc rbs sigma35dt-1C3, Psigma16 RGdT-1C3, Psigma35 RGdT-1C3, Pbadtigg-1C3, Pbad-rbssigma16dt 1C3, Pbad rbs sigma35-1C3, Pctrigg-pcTH1C3)
PCR (Pc sigma 16-dterm NG, Psigma11rgdt-3k3 ok, PbadtriggsT-1C3 no band, Pc anti35-dt , Pc anti16-dT ok, Pconst trigger-Pconst TH gfp NG)
Transformation( ligation product, I13458(pcAraC))
Preculture (Pc rbs anti16, Pc rbsanti35, Pc sigma11 RGdt-3k3, J23101, JM109)
Day 3-10/05/16(Sakata,Nasuda)
PCR (rbs16-1C3 ok, rbs35-1c3ok, Pc rbs sigma16dt-iC3ok, Pc rbs sigma35dt-1C3ok, Psigma16 RGdT-1C3ok, Psigma35 RGdT-1C3ok, Pbadtigg-1C3 NG, Pbad-rbssigma16dt 1C3 NG, Pctrigg-pcTH1C3NG)
Digestion (PRGdT 3k , Psigma16RGdt, Psigm35 RGdt, Pcanti16, Pcanti35, rbssigma35, Pc sigma16dt, Psigma16 RGdt, Pc sigma35dt, Pc sigma35RGdT, Pc THgfplva, Ptriggdt, Psigma11 RGdt, E18)
Gel Extraction File:T--UT-Tokyo-Labnote--1005
Ligation (Psigma16 RGdT-3K3, Psigma35RGdt-3K3, Pc anti16-1c3, Pc anti35-1C3, Pc sigma16-Psigma16 RGdt-3k3, Pc sigma35-Psigma35 RGdt-3k3, Pbad-rbs sigma35-1C3, PcThgfplva-3k3, PcTHgfplva-Pc Trigg-3k3, Psigma11-E18-3k3, Psigma16-THgfp-1C3 Transformation (ligation product + I20260 3k3)
Preculture (Psigma16 RGdt-1C3, Psigma 35 1c3, Pc sigma16-1c3, Pc sigma35-1C3, Pbad rbs sigma16 1C3, Pnrd gfp dterm 1C3, Pnrd gfplva dterm 1c3, Pctriggdt-pcTHgfplva-1c3, I20260) x3 check to see fluorescence the next day since PCR result wasn't too clear
Day 4-10/06/16(Sakata,Hamakawa,Sanada)
Miniprep
Glycerol stock
Digestion (I20260 -3k3, Pnrd gfp-3k3, Pc sigma 1c3, Psigma16 RGdt, Pbad sigma16 , Pc trigggdt Pc TH gfplva, Psigma 16, rbs sigma16, Pc AraC, Pbad trigg)
Gel extraction File:T--UT-Tokyo-Labnote--1006
PCR (Psigma16 RGdT-3K3, Psigma35RGdt-3K3, Pc anti16-1c3, Pc anti35-1C3, Pc sigma16-Psigma16 RGdt-3k3, Pc sigma35-Psigma35 RGdt-3k3, Pbad-rbs sigma35-1C3, PcThgfplva-3k3, PcTHgfplva-Pc Trigg-3k3, Psigma11-E18-3k3, Psigma16-THgfp-1C3)
Preculture (Psigma16 RGdT-3K3, Psigma35RGdt-3K3, Pc anti16-1c3, Pc anti35-1C3, Pc sigma16-Psigma16 RGdt-3k3, Pc sigma35-Psigma35 RGdt-3k3, Pbad-rbs sigma35-1C3, PcThgfplva-3k3, PcTHgfplva-Pc Trigg-3k3, Psigma11-E18-3k3, Psigma16-THgfp-1C3)
Day 5-10/07/16(Sakata,Narita)
No available result.
