McGill/Team 2: Repressilator

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Revision as of 18:54, 8 July 2007

May 2007
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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):

Amp: 1uL/mL
Kan: 10uL/mL
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.

I-brick from yesterday's midi prep
I-brick from yesterday's midid prep (2)
RSEI
RSE2
pEGFP mini prepped
pTet mini prepped
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

Performed a restriction digest of midi preps: pEGFP, pTet, J brick. pEGFP (EcoRI & XbaI); PTet (Xba1); J brick (EcoRV & Pst)
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
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.

Transformed plates again along with Supplemental plates.
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

Made 23 Kan+Amp plates.
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

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).

I5610 was transformed into commercial STBLE 3 cells with a HS of 45s and plated onto AMP resistant plates.
Seedings were preformed in duplicate for the Il5004 (20uL and 100uL plates) and the J40001 (100uL plates) and placed in the IS overnight.

June 12

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.
Made KAN (10uL per mL) and Amp/Kan plates
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.
Seedings of the Il5004 and Jh0001 were diluted to 100mL (100uL of DNA) for midiprep tomorrow.
Elowitz MC4100 cells were plated on antibiotic-free plates.

June 13

Il5004 and J40001 midiprep
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.
The new Elowitz cells (MC4100) for the repressilator were seeded for the chemically competent cell procedure to be conducted tomorrow.
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

I5610 were re-transformed into commercial STABLE3 cells as there was limited growth on the previous plates from before.
I5610 seeding were diluted to 100mL for the cc procedure.
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

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

June 18

More Kan plates and AMP/KAN plates were made using the lower concentration (5uL per mL) of KAN.
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.
Il5610 were seeded and placed in the IS overnight.

June 19

Last night's transformed plates of pGFP+MC4100 showed that the best heat shock time for MC4100 cells is 2 min. compared to 30s. as nothing grew at 30s. but good colony growth at 2 min.

Double transformation of the Il5004 and J40001 into commercial STBLE 3 cells using 2.5uL each of DNA for the I and J bricks with a 2 minute heat shock. Plated on new AMP/KAN resistant plates.
The seedings from yesterday were miniprepped and stored in the freezer.
Seeding of the pGFP in MC4100 for microscope verification tomorrow using supplemented Minimal Medium (as LB auto fluoresces) and of the Il5004 in commercial STABLE3 using LB.

June 20

Last night's double transformation of I15004 & J40001 were done using the wrong cells, with MC4100 the appropriate ones compared to STBLE3. This is as STBLE3 are not expression cells and BL21 cells are also inappropriate as they do not properly express Lac- properly.

Diluted pGFP seedings 20-fold upto 40ml. with supplemented Minimal Medium. These seedings were then viewed on the confocal microscope, showing excellent visual green fluorescence.
Diluted I15004 seedings to 100ml for Midiprep tomorrow.
A double transformation was performed again of I+J from left over midiprep DNA with MC4100 cells this time. Heat shock time used=2 min.
A restriction digest was performed on previously miniprepped I5610 using a double digest from EcoRV and HindIII restriction enzymes. However, after running a gel, a clear insertion was not seen in the cut plasmids. Will need to repeat procedure.

June 21

Unfortunately, the transformation from yesterday was accidently plated on AMP plates rather than AMP/KAN plates so the transformation was wasted. Colonies present on these plates indicate, however, that the J40001 was sucessfully transformed into the MC4100 cells.

A midi prep of the diluted I15004 from the STBLE3 was preformed and stored in the freezer for a digest tomorrow.
A double Transformation was repeated again for the I15004 and J40001 bricks from the miniprep DNA (16-06-07) using 2.5uL each of the DNA and a heat shock of 2 minutes into MC4100 cells. The transformed product was then properly plated on AMP/KAN plates and left in the incubator overnight.
The miniprepped I5610 was repeated using multiple cuts and with uncut plasmids and further run on a gel. the gel revealed strange cuts with the uncut DNA showing two bands, one characteristic of the plasmid and the other of the insert and the cuts themselves were unpredicted but one.

June 22

Unfortunately, there was no growth on the transformed plates again most likely due to inadequate amount of DNA (namely the I15004) or possibly poor plates.

Restriction Digest of the I15004 from yesterday's midi prep was preformed using three different cuts and run beside the uncut plasmid.
The restriction digest of the I5610 was also repeated using the same cuts and uncut plasmids and an additional single cut to linearize the plasmid to reduce supercoiling.
Again, a double transformation was preformed of the I15004 and J40001 into the MC4100 cells. 3.0uL of miniprepped (16-06-07) J40001 and 4.0uL of midiprepped (21-06-07) I15004 were transformed, HS for 2 minutes and plated on AMP/KAN plates.

Gels worked. On I15004 showed that the insert was present and that cuts were made by restriction enzymes.
On the Repressilator gel, different uncut bands showed different migrations on the gel. We concluded that this was due to supercoiling. Expected cuts were present on some of the restriction enzyme combinations.

June 26

A single I+J double transformed colony grew on the Amp/Kan plates. Due to the scarcity of colonies, half of the colony was spread on a new Amp/Kan plate and left in 37C incubation overnight, to promote further colony growth.
The other half was seeded in supplemented Minimal Medium and left in the S/I overnight. This will be diluted and viewed on the microscope for fluorescence tomorrow.

June 27

I+J seedings were diluted into 4 culture tubes (250ul each) in supplemented Minimal Medium.
Spreading of I+J colony on Amp/Kan plate was done incorrectly, as there were no single isolated colonies. Two new Amp/Kan plates were spread, one from the original I+J transformation plate and the other from a colony on the incorrectly spread plate.
J40001 was transformed on MC4100 cells to produce a negative control.<p>
The I+J dilutions, once reaching an O.D. of 0.25-0.30, were viewed at different cell densities (concentrations of Minimal Medium). IPTG (Lac- analog) was first added, left in incubation for 1hr, then washed several times with centrifugation in Minimal Medium. This produced 2 epitubes of I+J (One 4X concentrated, the other, 4x less concentrated).

The cells were then viewed on slides (fixed in 2% low grade Agarose) on the microscope and pictures were taken every 5 minutes for 1 hour.

Results: Images were continuously moving out of focus on the microscope, so oscillations were difficult to identify. After adjusting the focus before each image, due to the subjective nature of the manual focusing, images were still not sufficient to tell oscillations, although there were plenty of fluorescence.
The cells were then viewed on the confocal microscope. Images were taken, but again the microscope continually went out of focus. The microscope was left on for 4 hours, but due to the off-focusing, images were not sufficient to prove oscillations occurred.

June 28

Transformation of J40001 with MC4100 cells was successful on Amp plates.
Spreading of I+J colonies on new Amp/Kan plates were also successful, with plenty of available colonies to seed from.
Adam showed the procedure to correctly perform a PCR.
J40001 (negative control) was seeded in supplemental Minimal Medium.
I5610 (3ul) from Midiprep DNA was transformed (as a control) with MC4100 onto Amp plates.
I+J colonies from the newly spread plates were seeded again in supplemented minimal medium. 3 colonies were chosen an were left in the S/I overnight.
The I+J diluted cells were again placed under the microscope, this time using a lesser volume of cells & 4% low grade Agarose fixative, to hold the cells in place more strongly and to allow images to be taken from a single strong fluorescence focal point.

Results:Imaging was less off-focus than last time, but there were still some unusual bright and dim images. We concluded that this could be possibly due to photobleaching. Imaging also showed the presence of multiple layers on our microscopy sample.

July 6

Imaging on the MNI confocal was not successful because the sample kept drifting out of focus (or is it the microscope that can't keep focus?).
Imaging on the 5th floor confocal also showed that the focus was changing under bright field. The sample was not showing any fluorescence, though (bad colony?).
On Monday, we need to determine if it is the microscope or the sample that is causing the problem. We need some fixed bacterial slides to look at.

 Cloning Strategy for I15004 in pSB1AK3

A. Vector Preparation

1. Transform psb1AK3 in OneShot CcdB resistant cells

2. Plate on Streptomycin plates

3. Miniprep

4. Digest with EcoRI and PstI

B. Insert Preparation

1. PCR I15004 gene with standar biobrick primers

Primers: Forward: 5’ attaccgcctttgagtgagc 3’
        Reverse: 5’ tgccacctgacgtctaagaa 3’

2. PCR Purification

3. Digest with EcoRI and PstI

C. Ligation

1. Prepare an insert:vector ratio of ~1:3

2. Perform Ligation

D. Transformation

1. Into MC4100 cells (with J brick too)

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