Team:Groningen/Notebook/22 August 2009

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* From each tube 4mL of culture was collected in one 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
* From each tube 4mL of culture was collected in one 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
* Plasmids were eluted with 40μL MQ and stored in the fridge
* Plasmids were eluted with 40μL MQ and stored in the fridge
 +
 +
'''New over night cultures (2 days)'''
 +
 +
The six plates containing colonies of E.coli TOP10 with BBa_J61002-pArsR<sup>+</sup>/pZntR<sup>+</sup>/pCueO<sup>+</sup> were used to inoculate four tubes with 5 mL LB-amp<sub>100</sub> for each promoter.
 +
 +
:→ Instead of an over night incubation, the incubation time was increased to two nights, because it was sunday.
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'''Buoyancy Test'''
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 +
Four tubes with 6mL LB-amp<sub>100</sub> were inoculated with E.coli TOP10 pNL29, pSB1AC3-M/L-GVP, and pSB1AC3-RFP yesterday afternoon. All tubes had cell growth of comparable size, but no OD<sub>600</sub> measurement was made.
 +
 +
:→ The tubes were placed in a foam support to let the cells come to rest and placed at room temperature (a picture was taken as time zero, see below).
===Transporters===
===Transporters===
===Metal Accumulation===
===Metal Accumulation===
 +
*'''Transform ''E. coli'' with pSB1AC3-fMT, MymT, SmtA ligations'''
 +
** Use chemically competent cells and transform using heat shock (37&deg;C)
 +
**Transform ligation mixtures of
 +
::pSB1AC3 + MymT
 +
::pSB1AC3 + SmtA
 +
::pSB1AC3 + fMT
 +
::pSB1AC3 (self ligation control)
 +
::pSB1AC3-H + RFP (31-07-09; positive control)
 +
::MQ (negative control)
 +
**Plate on LBAmp and put o/n in 37&deg;C
===Vectors===
===Vectors===

Latest revision as of 22:41, 22 August 2009

Igemhomelogo.png

Wet

GVP Cluster

DONE Isolate plasmids from E.coli Top10 BBa_J23101 cells
DONE Plate E.coli Top10 BBa_J23101 cells from o.n. culture
TODO Pellet remaining cells for short storage in -20 box (not needed)
DONE Place the four test cultures with and without GVP on table at room temperature (make foto)
DONE Look at plates stored at 37C with BBa_J61002-pArsR+/pZntR+/pCueO+ and use colonies to grow o.n. cultures for plasmid isolation


Plates

Showed single colony growth on plates with J61002-pMEtal+RBS-RFP plasmids, and were stored in the fridge for future preculture growth.

→ The plates with high and low concentration of transformed cells showed colonies in the expected ratio.
→ On the plates for pZntR+ and pCueO+ no colonies were dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed.
→ The plates with pArsR+ plasmid showed only a few non-coloured colonies, mostly medium red colour, andn a few dark red colonies. It was already thought that the pArsR is a bit leaky and causes expression of RFP.

Over Night Cultures

→ Both cultures of BBa_J61002-J23101 showed growth, and can be used for plasmid isolation.
www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids BBa_J61002 with BBa_J23101 constitutive promoter with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture was collected in one 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 40μL MQ and stored in the fridge

New over night cultures (2 days)

The six plates containing colonies of E.coli TOP10 with BBa_J61002-pArsR+/pZntR+/pCueO+ were used to inoculate four tubes with 5 mL LB-amp100 for each promoter.

→ Instead of an over night incubation, the incubation time was increased to two nights, because it was sunday.

Buoyancy Test

Four tubes with 6mL LB-amp100 were inoculated with E.coli TOP10 pNL29, pSB1AC3-M/L-GVP, and pSB1AC3-RFP yesterday afternoon. All tubes had cell growth of comparable size, but no OD600 measurement was made.

→ The tubes were placed in a foam support to let the cells come to rest and placed at room temperature (a picture was taken as time zero, see below).

Transporters

Metal Accumulation

  • Transform E. coli with pSB1AC3-fMT, MymT, SmtA ligations
    • Use chemically competent cells and transform using heat shock (37°C)
    • Transform ligation mixtures of
pSB1AC3 + MymT
pSB1AC3 + SmtA
pSB1AC3 + fMT
pSB1AC3 (self ligation control)
pSB1AC3-H + RFP (31-07-09; positive control)
MQ (negative control)
    • Plate on LBAmp and put o/n in 37°C

Vectors

Dry

April
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
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4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30