From 2007.igem.org

yesterday -- tomorrow

DGAT biobrick: PCR

• Material:
  

- DGAT deltaPst-1: 7µL (35ng)
- Tampon: 10µL
- dNTP: 1µL (final 200µmol each)
- Taq: 0.5µL
- Water: 29µL
- Primer1: ForDGATbb: 2.5µL (final 0.5µM)
- Primer2: RevDGATbb: 2.5µL (final 0.5µM)

• PCR Program:
  

35 cycles:
- 95°C: 5min
- 95°C: 1min (X35)
- 55°C: 30s (X35)
- 72°C: 2min (X35)
- 72°C: 10 min
- 4°C: inf

• Quantity: 11ng/µL
  
• Expected size: 1443 bp

• After purification:
  

2µL of purification product (2µL of ladder)

Growth kinetics of w121 strain

We make an array to test growth of w121 on different growth media (LB, S0.2, S0.4, S0.6, S0.8), supplemented with different amounts of DAP.

Two questions are addressed by the following assay:

-What is the growth behaviour of w121 (dapA- strain) at different concentrations of DAP? -How does w121 strain grow on filtrates of MG1655 growth medium; that is, does MG1655 secrete DAP during growth?

MG1655 was grown on LB medium and the growth medium was filtered free of bacteria at different DO (Optical Densities) during exponential growth phase:

S0.2 (at DO=0.2) S0.4 (at DO=0.4) S0.6 S0.8

W121 was grown on different media & Growth kinetics were measured:

LB line B in the array S0.2 (at DO=0.2) line C in the array S0.4 line D in the array S0.6 line E & F in the array S0.8 line G in the array

In the different columns, DAP was added to the indicated final concentrations (without taking into account DAP produced by MG1655 regarding the recycled growth media)

Kinetic Array :w121 kinetic as a function of DAP and supplemented medium (S0.x)
	1	2	3	4	5	6	7	8	9	10	11	12
A	H20	H20	H20	H20	H20	H20	H20	H20	H20	H20	H20	H20
B	H20	LB+0µM DAP	LB+20µM DAP	LB+25µM DAP	LB+30µM DAP	LB+35µM DAP	LB+40µM DAP	LB+45µM DAP	LB+50µM DAP	LB+55µM DAP	LB+60µM DAP	H20
C	H20	S0.2+0µM DAP	S0.2+5µM DAP	S0.2+10µM DAP	S0.2+15µM DAP	S0.2+20µM DAP	S0.2+25µM DAP	S0.2+30µM DAP	S0.2+35µM DAP	S0.2+40µM DAP	S0.2+45µM DAP	H20
D	H20	S0.4+0µM DAP	S0.4+5µM DAP	S0.4+10µM DAP	S0.4+15µM DAP	S0.4+20µM DAP	S0.4+25µM DAP	S0.4+30µM DAP	S0.4+35µM DAP	S0.4+40µM DAP	S0.4+45µM DAP	H20
E	H20	S0.6+0µM DAP	S0.6+2µM DAP	S0.6+5µM DAP	S0.6+8µM DAP	S0.6+11µM DAP	S0.6+14µM DAP	S0.6+17µM DAP	S0.6+20µM DAP	S0.6+23µM DAP	S0.6+26µM DAP	H20
F	H20	S0.6+0µM DAP	S0.6+2µM DAP	S0.6+5µM DAP	S0.6+8µM DAP	S0.6+11µM DAP	S0.6+14µM DAP	S0.6+17µM DAP	S0.6+20µM DAP	S0.6+23µM DAP	S0.6+26µM DAP	H20
G	H20	S0.8+0µM DAP	S0.8+2µM DAP	S0.8+5µM DAP	S0.8+8µM DAP	S0.8+11µM DAP	S0.8+14µM DAP	S0.8+17µM DAP	S0.8+20µM DAP	S0.8+23µM DAP	S0.8+26µM DAP	H20
H	H20	H20	H20	H20	H20	H20	H20	H20	H20	H20	H20	H20

See results.

The 96 well plate is surrounded by H₂O in order to maintain humidity during the assay. In each slot, 200µL of the growth medium (LB or S0.x) is mixed with 2µL of w121 culture grown ON, and with different amount of DAP (see table). The growth profile is measured over a period of 30H, a DO measurement is acquired each 6min10s.