Team:GeorgiaTech/WeekFive

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8/29/2010
Results from the plates of 8.27.2010 transformation (transformed BL21 with pCOLD+mRFP):
non-IPTG plate- growth, many colonies, some are slightly red
IPTG plate- growth, many colonies, many are red, there are smaller red colones in the center along with larger normal ones; the last observation. Ryan suggested that it may indicate iptg stressed the cells in the middle (since IPTG could be more concentrated in middle).

What I did:
MAKING A STARTER Culture: I picked a slightly red colony from the non-iptg plate and innoculated 5 ml of LB+Carboamp with it.
Also, I put the iptg plate into the 15º C incubator and left it overnight

8/30/2010          Margo, Scott, Debika, Christian, and Christina
Observations: the non-iptg had more red colonies than it previously did on 8/29/2010

Induction experiment: inducing pCOLD+ mRFP under 4 different conditions (in BL21)


IPTG

-

-

+

+

15 degrees celsius

+

-

-

+

We added 1ml of starter solution to 50 ml of LB media and 50 ul of Carb. Put it in the 37C incubator shaker for an hour and a half. Checked OD levels after 1.5hrs for one flask. Since it was at .3, we checked again in 10 minutes.

After 10 minutes, the ODs values were as follows:

IPTG

-

-

+

+

15 degrees celsius

+

-

-

+

OD value

.51

.47

.47

.52

We wanted OD values to be @ ~0.4 (we got this number from the pCold manual). Added IPTG to one flask and put it in the 37C incubator w/ shaking along with another flask that had no IPTG.

Took the two other flasks and put it in the 15 degrees incubator shaker. After half an hour, we added IPTG to one of the flasks at 15 degrees, and left all 4 flasks in the respective incubators overnight. 

DMSO Stock (General protocol, applied here to pCOLD+mRFP containing cells from 8/30/2010)
Add to a cyrovial:
900 uL starter culture (from 8/30/2010 which was already fluorescent)
100 uL DMSO

  1. Vortex 5 seconds
  2. Put in -80c freezer (Gaucher lab)

Transformation of BL21 by pCOLD+mRFP (from 4º C and 15ºC cultures)

  1. Plasmid, cells should be on ice initially

1. 1 hour before transformation, spread 25 uL of 0.1 M IPTG onto one plate
2. + 1uL (1:1, ligation product) plasmid into 20 uL of competent cells (BL21) (edit:9.1.2010 the plasmid and cells should be on ice 15 mins before you start 2-8)
3. Sit on ice, 10 mins
4. place in 42ºC water bath, 30 seconds
5. place in ice, 2 minutes
6. +250 uL LB broth (edit 9/1/2010:  and wait 30 minutes @ 37C- tape down in incubator)
7. Plate: spread 100 uL onto plate, use spatula to spread around until resistance is felt.

    1. I plated an IPTG plate and a non-ITPG plate
    2. The IPTG plate had 25 uL of 0.1 M IPTG

8. Plates were left (upside down) in 37ºC incubator overnight, to be examined on Tues 31 Aug.
Note: Plate in incubator in room 218.

8/31/2010

Results from the four flasks incubated overnight:
+IPTG 37 C: Deep red cultures (color seen within an hour)
no IPTG 37 C: No color
+IPTG 15 C: Light red cultures (color seen the next morning)
no IPTG 15 C: No color
Contents of flasks bleached and discarded.

pCold in solution doesn’t require cold shock for expression, just IPTG. However, as seen from 8/29, pCold without IPTG colonies turned red when plated. Ryan noted that cells respond differently when in bulk vs. colonies, though these differences can’t really be generalized.

Results from plates from the transformation conducted on 8/30/2010:
No colonies were observed on the plates left in the incubator, cause: lack of incubation (essential for recovery after LB added) during transformation. During all transformations, the incubation step is crucial. Plates discarded.

Plan:
1. Make more media.
2. Make more plates.

 

Making LB media and plates
To make 1L LB (Lysogeny broth) medium:
5g Yeast abstract
10g Tryptone
5g NaCl
Fill up to 1L with dH2O.

To make solid plates, follow the above recipe and add 16g agar per 1L right before autoclaving.

Prepared 2L of LB media, 1L to be used as liquid broth and 1L to make plates.
Let cool at room temp.

For plate preparation:
Add 1:1000 antibiotic (CARB - plates marked with blue line) after autoclaving and cooling.
-1mL antibiotic per 1L media
Pipette 25mL into each plate.

Plan for 9/1/10:
1. Retransform from 15C colonies (not forgetting incubation!) from a miniprep.
2. Grow up cell cultures of pCOLD+mRFP run the following 4 conditions (as with the bulk solutions observed this morning):

IPTG

-

-

+

+

15 degrees celsius

+

- (37 c)

- (37 c)

+

Further, another colony will be plated without IPTG and left at room temp. This might allow us to determine if there are repeatable differences between plate and bulk bacteria behavior. The red color from warm, NON-IPTG plates leads us to think that we might see red without IPTG, and the extra room-temp control is determine if the response is temp-dependent.

9/1/2010
11 am: Prepared IPTG plate for transformation (25 uL IPTG) (Scott)
Margo, Scott, Mitesh, Gita, and Debika
12 pm: Performed another transformation following protocol from 08/30/10. We transformed pcold+mrfp (pcold+mrfp plasmid, the 15c one from 8.27.2010). This time, we put both the non-iptg plate and iptg plate into a 15c incubator.

9/2/2010
Results: no cells grew on either plate from 9/1/2010

3 pm: Gita and Christian
Objective: Determine if pCold is activated at intermediate concentrations of IPTG
Performed serial dilution of IPTG to make 1 mM, 10uM, and 100nM, 1mL stocks. Grew bacteria from DMSO stock starter-culture from 8/30 in test tubes and incubated in 37C and 15C shakers.

 

 

0 IPTG              (1 & 1’)

9.99e-6 M IPTG (2 & 2’)

9.99e-8 M IPTG (3 & 3’)

9.99e-10 M IPTG (4 & 4’)

37 C (#’)

 

 

 

 

15 C (#)