Team:TU Delft/9 August 2010 content

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-
=Alkane degradation=
+
=Lab work=
 +
==Alkane degradation==
-
==Digestion, Ligation, Transformation==
+
====Colony PCR====
 +
[https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=06_August_2010 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.
 +
 
 +
[[Image:TUDelft_20100809_PCR.jpg|450px]]
 +
 
 +
Lane description
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Primers'''
 +
|'''Expected length (bp)'''
 +
|'''✓'''
 +
|'''✗'''
 +
|-
 +
|L
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|n/a
 +
|-
 +
|1-8
 +
|013A
 +
|G00100 + G00101
 +
|1929
 +
|none
 +
|all
 +
|-
 +
|9-16
 +
|013K
 +
|G00100 + G00101
 +
|1929
 +
|none
 +
|all
 +
|-
 +
|L
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|n/a
 +
|}
 +
 
 +
Unfortunately none of the PCR products seemed to have the correct length. We'll try to make the BioBrick again.
 +
 
 +
====Digestion, Ligation, Transformation====
A digestion was done on a number of BioBricks for the production of new BioBricks.
A digestion was done on a number of BioBricks for the production of new BioBricks.
Line 53: Line 99:
The digestion was checked on a gel, together with the uncut BioBricks:
The digestion was checked on a gel, together with the uncut BioBricks:
 +
[[Image:TUDelft_20100809_digestion.jpg | 300px]]
-
'''Lane description'''
+
Lane description
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
Line 108: Line 155:
|}
|}
-
===Ligation===
+
For some samples a too low concentration was loaded, but furthermore the digestions looked ok.
 +
 
 +
 
 +
====Ligation====
Following the digestions, the fragments were [Team:TU_Delft/protocols/ligation ligated]] for 4 hours.
Following the digestions, the fragments were [Team:TU_Delft/protocols/ligation ligated]] for 4 hours.
Line 128: Line 178:
|}
|}
-
==Colony PCR==
+
==Emulsifier==
 +
Today we tried to see whether P. Putida excreeds an emulsifier. There were 3 samples:
 +
 
 +
* P. Putida grown over night on Glucose (OD: 1.559)
 +
* P. Putida grown over night on Octane (OD: 0.838)
 +
* M9 medium (negative control) (OD: 0.000)
 +
 
 +
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):
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''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
 +
|}
 +
 
 +
The conclusion is that with the additional of M9 or 4 M Urea the amount of hexane in the Tris buffer decreases. When the samples are used the emulsification becomes even lower. Probably due to the addition of the proteins the total amount of hydrophobic interfaces already increases. Therefore, a lower amount of hexane can be dissolved.

Latest revision as of 16:19, 15 August 2010

Contents

Lab work

Alkane degradation

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.

TUDelft 20100809 PCR.jpg

Lane description

# Description Primers Expected length (bp) ✓ ✗
L SmartLadder n/a n/a n/a n/a
1-8 013A G00100 + G00101 1929 none all
9-16 013K G00100 + G00101 1929 none all
L SmartLadder n/a n/a n/a n/a

Unfortunately none of the PCR products seemed to have the correct length. We'll try to make the BioBrick again.

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: TUDelft 20100809 digestion.jpg


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

For some samples a too low concentration was loaded, but furthermore the digestions looked ok.


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’

Emulsifier

Today we tried to see whether P. Putida excreeds an emulsifier. There were 3 samples:

  • P. Putida grown over night on Glucose (OD: 1.559)
  • P. Putida grown over night on Octane (OD: 0.838)
  • M9 medium (negative control) (OD: 0.000)

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

The conclusion is that with the additional of M9 or 4 M Urea the amount of hexane in the Tris buffer decreases. When the samples are used the emulsification becomes even lower. Probably due to the addition of the proteins the total amount of hydrophobic interfaces already increases. Therefore, a lower amount of hexane can be dissolved.

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