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

Day 3- 07/13/16

Transformations made on the previous day had failed.

Remade chloramphenicol plates after realising 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

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 igation 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 containated 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

Miniprep

Miniprep is done using the Wizard® Plus SV Minipreps DNA Purification System from Promega.

1. Add 1.5mL of overnight culture in a microcentrifuge tube
2. Centrifuge at 15000 rmp for 1min
3. Discard suspension
4. Add 1mL of overnight culture
5. Centrifuge at 15000 rmp for 1min
6. Discard suspension
7. Add 250uL of Cell Resuspension Solution
8. Vortex
9. Add 250uL of Cell Lysis Solution
10. Invert
11. Add 10uL of Alkaline Protease
12. Invert
13. Leave for 3min
14. Add 350uL of Neutralization solution
15. Centrifuge at 15000rmp for 10min
16. Place column in collection tube and transfer all suspension to the column
17. Centrifuge at 15000rmp for 1min
18. Discard flow-through
19. Add 750uL of Wash Solution
20. Centrifuge at 15000rmp for 1min
21. Add 250uL of Wash Solution
22. Set the column in a new microcentrifuge tube and discard the collection tube
23. Add 100uL of Nuclease-free water to the column
24. Leave for 1min
25. Centrifuge at 13000rmp for 1min