McGill/Team 2: Repressilator

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May 2007

May 11

Autoclaved LB, Agar and Minimal Medium. Made plates with AmpR, KanR and both Amp and Kan. Concentrations used (same for cell cultures):

1. Amp: 1uL/mL
2. Kan: 10uL/mL
3. Cam: 0.8uL/mL (from Elvis' aliquots)

The receipe for M9 Minimal Medium is in my lab notebook which is in the lab so I'll post it later under the Protocols section. Hopefully we can get the 2007 wiki soon. Horia

May 14

Moved plates from iGEM fridge to fridge on C block (end of hall) Made 100X Yeast extract and 10X Dextrose (for supplementing minimal medium) Prepared CaCl2 and CaCl2/Glycerol 10% solutions for CC procedure of tomorrow Will seed tonight. Horia

May 15

Performed cc procedure with top10 (elvis' home made), bl21 a1 and stble3

Performed seeding procedure on plates from last year: ILS004, duplicate copies of 5mL LB with 50uL KAN; RSE/J40001, duplicate copies of 5mL LB with 5uL AMP; Place in IS overnight

May 16

Transformed cc cells with pUC19 and plated

Repeated seeding procedure as that from previous day did not prove any results (defective shaker?). Both ILS004 and RSE repeated in triplicate with its respective antibiotic. Placed in IS at noon and two samples diluted with LB (100mL to RSE sample and 80mL to ILS004) and placed in the IS overnight. No cell growth.

May 17

Nothing grew on most of the plates. Only 2 colonies on a stble3 plate. Decided the cells were bad because 1. we did not keep them on ice while we transported them etc. and 2. some cells waited a long time on ice because we had to do sequential centrifugations (not enough of one kind of tube). Removed from incubator at night and placed in the fridge.

Susan, Avi and Jimmy seeded cells again for Friday. BL21, Top10 and STBLE3 repeated in triplicate in 5mL LB and placed in SI overnight.

May 18

Performed cc procedure.

May 22

Made LB for Seeding procedure from 15.5g of solid LB to 500ml of water - x3.

Performed transformation of competent cells with PGFP (which was supposed to be pUC19) (Top 10 and Stble 3) and BL21 with PGFP and Amp antibiotic to validate their quality.

For the transformation procedure, used LB instead of S.O.C. Medium, due to its unavailability, then spread the cells on Agar gel plates with Amp and LB and placed in incubator overnight.

Made some additional Agar gel plates with Amp, which were kept in the freezer for future use.

May 23

Test transformation results

BL21
30s / 2 min Heat Shock
20 µl = 0 cells / many cells
75 µl = 0 cells / many cells

Top 10
30s / 2 min Heat Shock
20 µl = many cells / many cells
75 µl = many cells / many cells

Stable 3
30s / 2 min Heat Shock
20 µl = ~ 30 cells / 7 cells
75 µl = ~ 30 cells / 3 cells

Then performed transformation using Top 10 cells. Added biobrick (10M well) with Kan plates (200µl & 50µl) and RSE with Amp plates (100µl & 20µl). Left in incubator overnight at 37C.

Also, six seeding were preformed on test colonies transfected with PGFP and let in the IS overnight.

May 24

Miniprep protocol (from iGEM 2006) of Top10 and STBLE3 cells transformed with pGFP plasmids and seeded yesterday. BL21 cells were tested for the presence of the pGFP plasmid through microscope examination which proved positive results.

A restriction digest was implemented on the isolated DNA from the Top10 and STBLE3 cells 1.5uL Buffer 2
0.5uL of BSA
3uL DNA
8uL water
1uL EcoRI
1uL XbaI

Due to lack of ethidium bromide, a gel could not be made so the run was postponed for Friday.

May 25

First, the Top10 and STBLE3 were again digested as per the digestion above to be run in parallel with the digestion from yesterday.

An electrophoresis gel was made 50mL of TAE
0.35g agarose
heated ~20s then cooled to <60 degrees Celsius
7uL of ethidium bromide was added and poured into a gel plate

1.74uL of 10X loading dye was added to the DNA and the gel was run for 45 minutes at 100V; upon completion, a picture was taken to reveal no bands.

A miniprep was also preformed on the iBrick (Il5004 plasmid) and RSE cells (J40001 plasmid) seeded the day before and DNA stored in the freezer.

May 28

Today, a restriction digest was preformed on the iBrick and RSE cells with the following respective solutions in duplicate

IL5004
1.5uL Buffer 3
0.5uL BSA
3uL DNA
1uL PvuII
1uL PstI
8uL water

Jh0001
1.5uL Buffer 3
0.5uL BSA
3uL DNA
1uL EcoRV
1uL PstI
8uL water

Again, a gel was run and again, a negative result was obtained showing no bands.

May 30

Midi prep of iBrick with a new midi prep kit.

Seeding of J-brick (RSE) and Jay's pEGFP and pTet

May 31

A gel was run with the following wells.

1. I-brick from yesterday's midi prep
2. I-brick from yesterday's midid prep (2)
3. RSEI
4. RSE2
5. pEGFP mini prepped
6. pTet mini prepped
7. Elvis's pGFP

The gel from yesterday's midi prep was successful and DNA was present.

Dilutions where implemented on the seedings from yesterday

June 2007

June 4

1. Performed a restriction digest of midi preps: pEGFP, pTet, J brick. pEGFP (EcoRI & XbaI); PTet (Xba1); J brick (EcoRV & Pst)
2. After digest, made a gel using L2 Ladder. To prepare L2 use 3:1:1 (H20:Dye:TAE).
   Wells: 1. pEGFP 2. pTet 3. J Brick
3. Seeded the following plates: I+J (20ul&100ul); I1500S (1x with LB & 1x with M.M.); J4000I (1x with LB & 1x with M.M.)

Gel showed abnormal bands with smudges. This could be due to improper well loading or gel transfer.

June 5

After seeding, results showed that the J brick grew abnormally, growing better with M.M. than with LB. I+J (double seedings) did not grow at all in both mediums. The I brick grew in LB but not with M.M.
Conclusion: Possibly bad colonies were selected or there were problems with the transformation procedure.

1. Transformed plates again along with Supplemental plates.
2. Seeded I, J and I+J (2007 & 2006 plates) again, 3 colonies selected each as well as 2 Control tubes (1x LB; 1x LB+Kan/AMP)

June 6

1. Made 23 Kan+Amp plates.
2. Transformed commercial biobricks (I15004, J40001, Elowitz (I5610)) in pSB1A2 plasmid (AmpR) with Stble 3 cells. Left overnight in incubation room.

June 7

Results from last night's transformation:
I15004
20ul: 3 big colonies, many small colonies
100ul: 20 big colonies, many small colonies
J40001
20ul: 2 big colonies, few small colonies
100ul: 7 big colonies, no small colonies
I5610
20ul: 1-2 small colonies, little growth
100ul: 1-2 small colonies, little growth

Seeded plates with LB. 1 colony from each plate chosen (2x each biobrick). 6 seedings in total. Left in S/I overnight.

June 8

Of last night's seedings, only two tubes showed visible growth: I15004(100ul)& J40001(100ul). No growth seen in I15610(Elowitz) in both concentrations.

Performed a Miniprep of I15004 & J40001. Kept in freezer.

June 11

1. Restriction Digest and Screening: I15004 and J40001 plasmids miniprepped on Friday were digested as per the following
   

I15004
1.5 uL Buffer 3
0.5uL BSA
4uL DNA
7uL water
1uL PVUII
1uL PstI

J40001
1.5 uL Buffer 3
0.5uL BSA
4uL DNA
7uL water
1uL EcoRV
1uL PstI

Digestion was run on gel though there were no bands as the TAE was improperly made. Seeding were repeated to be prepared tomorrow (2X I15004 20uL, 2X I15004 100ul, 2X J40001 100uL).

1. Transformation

I5610 was transformed into commercial STBLE 3 cells with a HS of 45s and plated onto AMP resistant plates.

1. Seeding

Seedings were preformed in duplicate for the Il5004 (20uL and 100uL plates) and the Jh0001 (100uL plates) and placed in the IS overnight.

June 12

1. Restriction Digest
   

Repeated the digest from June 11 and run on a properly made gel. Results indicated that the J brick was indeed present though the I brick was inconclusive due to erroneous cutting. Diluted uncut plasmids (1ul of DNA to 5uL of water) were also run and the I-brick indicated that the insert my not be present so further analysis is required. Also note that the bio-brick was diluted with 3uL of water to dissolve the dry DNA.

1. Made KAN (10uL per mL) and Amp/Kan plates

1. Seeding
   

Seeding of the I5610 in commercial STBLE 3 cells and placed in the IS overnight. Results were poor as the wrong STBLE 3 cells were used and not enough DNA was implemented due to the dilution preformed earlier.

1. Dilutions
   

Seedings of the Il5004 and Jh0001 were diluted to 100mL (100uL of DNA) for midiprep tomorrow.

1. Plating
   

Elowitz MC4100 cells were plated on antibiotic-free plates.

June 13

1. Midiprep
   

Il5004 and Jh0001 midiprep

1. Transformation
   

Re-transformation of the I5610 into the correct commercial STBLE3 cells using 2uL of DNA instead of 1.5uL as it the DNA was previously diluted. The transformed cells were plated on AMP resistent plates with volumes of 10, 50 and 100uL.

1. Seeding

The news Elowitz cells (MC4100) for the repressilator were seeded for the chemically competent cell procedure to be conducted tomorrow.

1. Make new Kan Antibiotics
   

New KAN antibiotics were made and sterilized as per the procedure recorded in the protocal section. New information: since Il5004 is a medium copy number plasmid, we should be using 5uL per ml opposed to 10uL per ml for plating and seeding with this DNA thus from now on, these concentrations are implemented.

June 14

1. Transformation
   

Il5610 were re-transformed into commercial STABLE3 cells as there was limited growth on the previous plates from before.

1. Dilution
   

Il5610 seeding were diluted to 100mL for the cc procedure.

1. Restriction Digest
   

Restriction digest of I and J plasmids from yesterday's midis as per the cuts on June 11. Again, the I brick yielded inconclusive cuts and smaller than expected total plasmids indicating that the insert may not be present; thus, a PCR will be preformed to confirm the presence of the insert and to amplify the DNA.

June 15

1. Primers
   

Research using primer 3 program to find unique and adequate primers to be ordered in due time.

June 18

1. Make Plates
   

More Kan plates and AMP/KAN plates were made using the lower concentration (5uL per mL) of KAN.

1. Transformation
   

The MC4100 cc cells were transformed with 3uL of pGFP using both a 30s and 2min HS and then plated on AMP plates. IL5004 (1.5uL) were also tranformed into STBLE3 using a 2min HS and plated on the new KAN plates. Results: The MC4100 cells only yielded adequate colonies for the 2min heat shock and even with this, a large volume (75-100uL on a plate) yielded a dozen colonies. For the Il5004, there again was only a few colonies (less than before) an with a large volume of plating mixture (125uL) thus indicating that a larger amount of DNA should be used especially considering that the bio-brick has been diluted.

1. Seeding
   

Il5610 were seeded and placed in the IS overnight.

June 19

1. Transformation
   

Double transformation of the Il5004 and Jh0001 into commercial STBLE 3 cells using 2.5uL each of DNA for the I and J bricks with a 2minute heat shock. Plated on new AMP/KAN resistant plates.

1. Miniprep
   

The seedings from yesterday were miniprepped and stored in the freezer.

1. Seeding
   

Seeding of the pGFP in MC4100 for microscope verification tomorrow and of the Il5004 in commercial STABLE3.