Team:TU Delft/9 August 2010 content

From 2010.igem.org

(Difference between revisions)
(→Alkane degradation)
(→Emulsifier)
Line 132: Line 132:
=Emulsifier=
=Emulsifier=
 +
Today we tried to see whether P. Putida excreeds an emulsifier. Therefore we used the following protocol:
 +
 +
Materials:
 +
*25 mM triethanolamine (TEA) buffer, pH 8
 +
*1% lysozyme in TEA
 +
*4M urea
 +
*10% streptomycin in TEA
 +
*Glass beads
 +
*25 mM Tris buffer (pH 8)
 +
 +
Method:
 +
*Harvest 25 mL bacterial cells by centrifugation at 10.000 rpm for 10 min
 +
*Collect the cell free supernatant and store it
 +
*Wash pellet with TEA buffer
 +
*Freeze pellet in -70 C for 15 min
 +
*Resuspend pellet 2 mL TEA + 40 ul 1% lysozyme (final concentration 0.02%)
 +
*Incubate for 10 min at RT
 +
*Disrupt cells with glass beads. Vortex for 10 minutes
 +
*Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in supernatant)
 +
*Aliquot 1.8 mL supernatant into fresh eppendorf tubes and add 200 ul 10% streptomycin in TEA (final concentration 1%)
 +
*Incubate 10 min at RT
 +
*Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in pellet)
 +
*Resuspend pellet in 4 M urea
 +
*Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in supernatant)
 +
 +
The samples were tested according to the emulsification assay protocol (@TODO: link):
 +
 +
{|
 +
|#
 +
|Buffer (ul)
 +
|Hexane (ul)
 +
|Sample (ul)
 +
|Absorbance (660nm)
 +
|-
 +
|1
 +
|950
 +
|50
 +
|
 +
|0.272
 +
|-
 +
|2
 +
|900
 +
|50
 +
|50 M9
 +
|0.141
 +
|-
 +
|3
 +
|950
 +
|0
 +
|50 M9
 +
|0.001
 +
|-
 +
|4
 +
|900
 +
|50
 +
|50 Glucose culture (sup)
 +
|0.100
 +
|-
 +
|5
 +
|950
 +
|0
 +
|50 Glucose culture (sup)
 +
|0.023
 +
|-
 +
|6
 +
|900
 +
|50
 +
|50 Octane culture (sup)
 +
|0.161
 +
|-
 +
|7
 +
|950
 +
|0
 +
|50 Octane culture (sup)
 +
|0.013
 +
|-
 +
|8
 +
|900
 +
|50
 +
|50 Glucose culture (pellet)
 +
|0.118
 +
|-
 +
|9
 +
|950
 +
|0
 +
|50 Glucose culture (pellet)
 +
|0.014
 +
|-
 +
|10
 +
|900
 +
|50
 +
|50 Octane culture (pellet)
 +
|0.168
 +
|-
 +
|11
 +
|950
 +
|0
 +
|50 Octane culture (pellet)
 +
|-0.002
 +
|-
 +
|12
 +
|900
 +
|50
 +
|50 4 M Urea
 +
|0.175
 +
|-
 +
|13
 +
|950
 +
|0
 +
|50 4 M Urea
 +
|-0.001
 +
|}

Revision as of 08:31, 10 August 2010

Contents

Alkane degradation

Digestion, Ligation, Transformation

A digestion was done on a number of BioBricks for the production of new BioBricks.

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 009A EcoRI SpeI PvuI 2 (Biolabs) ✓ ‘E - J61100 - rubA4 - S’
2 012K EcoRI XbaI ✗ 2 (Biolabs) ✓ ‘E – J61100 - rubR - B0015 – X’
3 017A SpeI PstI 2 (Biolabs) ✓ ‘P - pSB1A2 – J61100 - ladA – S’
4 018A XbaI PstI PvuI 3 (Biolabs) ✓ ‘X – J61107 - ALDH – P’

The digestion was checked on a gel, together with the uncut BioBricks:


Lane description

# Description Expected size (bp) OK? # Description Expected size (bp) OK?
L Smartladder (3μl) n/a n/a 5 017A cut
1 009A cut 6 017A uncut n/a
2 009A uncut n/a 7 018A cut
3 012K cut 8 018A uncut n/a
4 012K uncut n/a

Ligation

Following the digestions, the fragments were [Team:TU_Delft/protocols/ligation ligated]] for 4 hours.

# BioBrick Fragment 1 Recipient vector
1 013AK 10 μL ‘E – J61100 - rubA4 – S’ 10 μL ‘X – J61100 - rubR - B0015 - pSB1AK3 – E’
2 020A 10 μL ‘P - pSB1A2 – J61100 - ladA – S’ 10 μL ‘X – J61107 - ALDH – P’

Colony PCR

Friday, a colony PCR on colonies from a plate with transformants with BioBrick 013AK did not give positive results. But this doesn't mean that none of the colonies are correct. To check we did another colony PCR today on 8 more colonies of the AMP plate as well as the KAN plate. Hopefully a the PCR product of a colony will show the correct length.

Emulsifier

Today we tried to see whether P. Putida excreeds an emulsifier. Therefore we used the following protocol:

Materials:

  • 25 mM triethanolamine (TEA) buffer, pH 8
  • 1% lysozyme in TEA
  • 4M urea
  • 10% streptomycin in TEA
  • Glass beads
  • 25 mM Tris buffer (pH 8)

Method:

  • Harvest 25 mL bacterial cells by centrifugation at 10.000 rpm for 10 min
  • Collect the cell free supernatant and store it
  • Wash pellet with TEA buffer
  • Freeze pellet in -70 C for 15 min
  • Resuspend pellet 2 mL TEA + 40 ul 1% lysozyme (final concentration 0.02%)
  • Incubate for 10 min at RT
  • Disrupt cells with glass beads. Vortex for 10 minutes
  • Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in supernatant)
  • Aliquot 1.8 mL supernatant into fresh eppendorf tubes and add 200 ul 10% streptomycin in TEA (final concentration 1%)
  • Incubate 10 min at RT
  • Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in pellet)
  • Resuspend pellet in 4 M urea
  • Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in supernatant)

The samples were tested according to the emulsification assay protocol (@TODO: link):

# Buffer (ul) Hexane (ul) Sample (ul) Absorbance (660nm)
1 950 50 0.272
2 900 50 50 M9 0.141
3 950 0 50 M9 0.001
4 900 50 50 Glucose culture (sup) 0.100
5 950 0 50 Glucose culture (sup) 0.023
6 900 50 50 Octane culture (sup) 0.161
7 950 0 50 Octane culture (sup) 0.013
8 900 50 50 Glucose culture (pellet) 0.118
9 950 0 50 Glucose culture (pellet) 0.014
10 900 50 50 Octane culture (pellet) 0.168
11 950 0 50 Octane culture (pellet)
12 900 50 50 4 M Urea 0.175
13 950 0 50 4 M Urea
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