Team:TU Delft/6 July 2010 content

From 2010.igem.org

(Difference between revisions)
(Lab work)
 
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-
==Lab work==
+
=Lab work=
-
<h4>Ordered DNA stocks</h4>
+
==Ordered DNA stocks==
The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!
The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!
Line 23: Line 23:
* PhPFD-β (Ampicillin)
* PhPFD-β (Ampicillin)
-
<h4>Characterization of Anderson RBS sequences</h4>
+
==Characterization of Anderson RBS sequences==
Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]]:
Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]]:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
-
|'''Digestion reaction'''
+
|'''Sample'''
-
|'''Used Buffer'''
+
|''' Enzyme 1'''
 +
|'''Enzyme 2'''
 +
|'''Buffer'''
 +
|'''BSA'''
|'''Needed fragment'''
|'''Needed fragment'''
|-
|-
|C1
|C1
-
|1.15 μg J61100 + SpeI + PstI
+
|1.15 μg J61100  
-
|Buffer 2 (BioLabs)
+
|SpeI
-
|‘S – J61100 - pSB1A2 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|
 +
|‘S–J61100 pSB1A2–P’
|-
|-
|C2
|C2
-
|1.68 μg J61101 + SpeI + PstI
+
|1.68 μg J61101  
-
|Buffer 2 (BioLabs)
+
|SpeI
-
|‘S – J61101 - pSB1A2 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|
 +
|‘S–J61101 pSB1A2–P’
|-
|-
|C3
|C3
-
|1.30 μg J61107 + SpeI + PstI
+
|1.30 μg J61107  
-
|Buffer 2 (BioLabs)
+
|SpeI
-
|‘S – J61107 - pSB1A2 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|
 +
|‘S–J61107 pSB1A2–P’
|-
|-
|C4
|C4
-
|0.95 μg J61117 + SpeI + PstI
+
|0.95 μg J61117  
-
|Buffer 2 (BioLabs)
+
|SpeI
-
|‘S – J61117 - pSB1A2 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|
 +
|‘S–J61117 pSB1A2–P’
|-
|-
|C5
|C5
-
|0.51 μg J61127 + SpeI + PstI
+
|0.51 μg J61127
-
|Buffer 2 (BioLabs)
+
|SpeI
-
|‘S – J61127 - pSB1A2 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|
 +
|‘S–J61127 pSB1A2–P’
|-
|-
|D1
|D1
-
|2.59 μg I13401 + XbaI + PstI
+
|2.59 μg I13401  
-
|Buffer 2 (BioLabs)
+
|XbaI
-
|‘X – I13401 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|
 +
|‘X–I13401–P’
|-
|-
|D2
|D2
-
|2.65 μg I13401 + XbaI + PstI
+
|2.65 μg I13401
-
|Buffer 2 (BioLabs)
+
|XbaI
-
|‘X – I13401 – P’
+
|PstI
-
|-
+
|2 (BioLabs)
 +
|
 +
|‘X–I13401–P’
|}  
|}  
-
The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a 1% agarose gel for comparison with the non-digested BioBricks:
+
The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]] for comparison with the non-digested BioBricks:
-
[[Image:TUDelft_undigested-digested_06_July.png|500px|thumb|left|Digestion check]]
+
[[Image:TUDelft_undigested-digested_06_July.png|500px|thumb|left|1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 1 μL sample + 4 μL MQ + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker]]
Lane description:
Lane description:
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|'''Description'''
|'''Description'''
|'''Expected Length (bp)'''
|'''Expected Length (bp)'''
-
|'''Remarks'''
+
|'''Status'''
|-
|-
-
|1
+
|M1
|SmartLadder marker (5 μL)
|SmartLadder marker (5 μL)
|n/a
|n/a
-
|
+
|n/a
|-
|-
-
|2
+
|1
-
|Undigested J61100 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61100 in pSB1A2
-
|
+
|2134
-
|Circular Plasmid
+
|<font color=limegreen>✓</font>
|-
|-
|3
|3
-
|Undigested J61101 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61101 in pSB1A2
-
|
+
|2134
-
|Circular Plasmid
+
|<font color=limegreen>✓</font>
|-
|-
|4
|4
-
|‘S – J61100 - pSB1A2 – P’
+
|J61100 + SpeI + PstI 
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>✓</font>
|-
|-
|5
|5
-
|‘S – J61101 - pSB1A2 – P’
+
|J61101 + SpeI + PstI 
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>✓</font>
|-
|-
|6
|6
-
|Undigested J61107 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61107 in pSB1A2
-
|
+
|2134
-
|Circular plasmid
+
|<font color=limegreen>✓</font>
|-
|-
|7
|7
-
|‘S – J61107 - pSB1A2 – P’
+
|J61107 + SpeI + PstI
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>✓</font>
|-
|-
|8
|8
-
|Undigested J61117 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61117 in pSB1A2
-
|
+
|2134
-
|Circular plasmid
+
|<font color=limegreen>✓</font>
|-
|-
|9
|9
-
|‘S – J61117 - pSB1A2 – P’
+
|J61117 + SpeI + PstI 
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>✓</font>
|-
|-
|10
|10
-
|Undigested J61127 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested J61127 in pSB1A2
-
|
+
|2134
-
|Circular plasmid
+
|<font color=limegreen>✓</font>
|-
|-
|11
|11
-
|‘S – J61127 - pSB1A2 – P’
+
|J61127 + SpeI + PstI
-
|2116
+
|18, 2116
-
|Visible
+
|<font color=limegreen>✓</font>
|-
|-
|12
|12
-
|Undigested I13401 (1 μL + 4 μL MQ + 1 μL Loading Buffer)
+
|Undigested I13401 in pSB1A2
-
|
+
|2936
-
|Circular plasmid
+
|<font color=limegreen>✓</font>
|-
|-
|13
|13
-
|‘X – I13401 – P’
+
|I13401 + XbaI + PstI
-
|883
+
|883, 2053
-
|Visible
+
|<font color=limegreen>✓</font>
|-
|-
|14
|14
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|BioRad EZ Load
|BioRad EZ Load
|n/a
|n/a
-
|Visible
+
|n/a
|}
|}
-
Subsequent over night [http://2010.igem.org/Team:TU_Delft/protocols/ligation ligation] yielded:
+
Followed by over night [http://2010.igem.org/Team:TU_Delft/protocols/ligation ligation]:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''BioBrick'''
|'''BioBrick'''
|'''Recipient plasmid'''
|'''Recipient plasmid'''
-
|'''Fragment insert'''
+
|'''Fragment'''
|'''Final volume'''
|'''Final volume'''
|-
|-
|1
|1
|K398500A
|K398500A
-
|130 μg ‘S – J61100 - pSB1A2 – P’
+
|130 μg ‘S–J61100 pSB1A2–P’
|154 μg ‘X-I13401-P’  
|154 μg ‘X-I13401-P’  
|26 μL
|26 μL
Line 173: Line 196:
|2
|2
|K398501A
|K398501A
-
|206 μg ‘S – J61101 - pSB1A2 – P’
+
|206 μg ‘S–J61101 pSB1A2–P’
|247 μg ‘X-I13401-P’  
|247 μg ‘X-I13401-P’  
|25 μL
|25 μL
Line 179: Line 202:
|3
|3
|K398502A
|K398502A
-
|196 μg ‘S – J61107 - pSB1A2 – P’
+
|196 μg ‘S–J61107 pSB1A2–P’
|247 μg ‘X-I13401-P’
|247 μg ‘X-I13401-P’
|25 μL
|25 μL
Line 185: Line 208:
|4
|4
|K398503A
|K398503A
-
|201 μg ‘S – J61117 - pSB1A2 – P’
+
|201 μg ‘S–J61117 pSB1A2–P’
|247 μg ‘X-I13401-P’
|247 μg ‘X-I13401-P’
|25 μL
|25 μL
Line 191: Line 214:
|5
|5
|K398504A
|K398504A
-
|202 μg ‘S – J61127 - pSB1A2 – P’
+
|202 μg ‘S–J61127 pSB1A2–P’
|247 μg ‘X-I13401-P’
|247 μg ‘X-I13401-P’
|25.5 μL
|25.5 μL
|-
|-
|6
|6
-
|Ligase Control (J61117 - pSB1A2)
+
|negative control
-
|123 μg ‘S – J61117 - pSB1A2 – P’
+
|123 μg ‘S–J61117 pSB1A2–P’
-
|None
+
|'''-'''
|15 μL
|15 μL
|-
|-
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To all samples one-tenth of the total volume ligase buffer was added.
To all samples one-tenth of the total volume ligase buffer was added.
-
<h4>Emulsifier</h4>
+
==Emulsifier==
Pieter is testing different condition for the emulsifying Assay. To determine the emulsifying activity we set up a calibration curve with SDS. He prepared the following samples:  
Pieter is testing different condition for the emulsifying Assay. To determine the emulsifying activity we set up a calibration curve with SDS. He prepared the following samples:  
Line 245: Line 268:
|}
|}
-
==Kampioenen!!!!==
+
=Kampioenen!!!!=
Today the Dutch team will play the semi-final of the World Cup!
Today the Dutch team will play the semi-final of the World Cup!

Latest revision as of 19:23, 2 August 2010

Contents

Lab work

Ordered DNA stocks

The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!

We were so excited; we immediately dissolved the DNA in water and performed a transformation with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.

  • AlkB2 (Ampicillin)
  • rubA3 (Ampicillin)
  • rubA4 (Ampicillin)
  • rubB (Kanamycin)
  • ladA (Ampicillin)
  • ADH (Ampicillin)
  • ALDH (Kanamycin)
  • bbc1 (Ampicillin)
  • AlnA (Ampicillin)
  • OprG (Ampicillin)
  • PalkS1-2 (Ampicillin)
  • PalkB (Ampicillin)
  • P(CaiF) (Ampenicillin)
  • AlkS (Kanamycin)
  • PhPFD-α (Ampicillin)
  • PhPFD-β (Ampicillin)

Characterization of Anderson RBS sequences

Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were digested:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
C1 1.15 μg J61100 SpeI PstI 2 (BioLabs) ‘S–J61100 pSB1A2–P’
C2 1.68 μg J61101 SpeI PstI 2 (BioLabs) ‘S–J61101 pSB1A2–P’
C3 1.30 μg J61107 SpeI PstI 2 (BioLabs) ‘S–J61107 pSB1A2–P’
C4 0.95 μg J61117 SpeI PstI 2 (BioLabs) ‘S–J61117 pSB1A2–P’
C5 0.51 μg J61127 SpeI PstI 2 (BioLabs) ‘S–J61127 pSB1A2–P’
D1 2.59 μg I13401 XbaI PstI 2 (BioLabs) ‘X–I13401–P’
D2 2.65 μg I13401 XbaI PstI 2 (BioLabs) ‘X–I13401–P’

The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a 1% agarose gel for comparison with the non-digested BioBricks:

1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 1 μL sample + 4 μL MQ + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker

Lane description:

# Description Expected Length (bp) Status
M1 SmartLadder marker (5 μL) n/a n/a
1 Undigested J61100 in pSB1A2 2134
3 Undigested J61101 in pSB1A2 2134
4 J61100 + SpeI + PstI 18, 2116
5 J61101 + SpeI + PstI 18, 2116
6 Undigested J61107 in pSB1A2 2134
7 J61107 + SpeI + PstI 18, 2116
8 Undigested J61117 in pSB1A2 2134
9 J61117 + SpeI + PstI 18, 2116
10 Undigested J61127 in pSB1A2 2134
11 J61127 + SpeI + PstI 18, 2116
12 Undigested I13401 in pSB1A2 2936
13 I13401 + XbaI + PstI 883, 2053
14 empty empty empty
15 BioRad EZ Load n/a n/a

Followed by over night ligation:

# BioBrick Recipient plasmid Fragment Final volume
1 K398500A 130 μg ‘S–J61100 pSB1A2–P’ 154 μg ‘X-I13401-P’ 26 μL
2 K398501A 206 μg ‘S–J61101 pSB1A2–P’ 247 μg ‘X-I13401-P’ 25 μL
3 K398502A 196 μg ‘S–J61107 pSB1A2–P’ 247 μg ‘X-I13401-P’ 25 μL
4 K398503A 201 μg ‘S–J61117 pSB1A2–P’ 247 μg ‘X-I13401-P’ 25 μL
5 K398504A 202 μg ‘S–J61127 pSB1A2–P’ 247 μg ‘X-I13401-P’ 25.5 μL
6 negative control 123 μg ‘S–J61117 pSB1A2–P’ - 15 μL

To all samples one-tenth of the total volume ligase buffer was added.

Emulsifier

Pieter is testing different condition for the emulsifying Assay. To determine the emulsifying activity we set up a calibration curve with SDS. He prepared the following samples:

# Hexane (mL) Tris Buffer pH 8 (mL) 10% SDS (mL)
B 1 1.1 0
100 1 1 0.1
50 1 1.05 0.05
25 1 1.075 0.025
10 1 1.09 0.01
5 1 1.095 0.005

Kampioenen!!!!

Today the Dutch team will play the semi-final of the World Cup! Hear us cheer! Stop sound

Pieter by the BBQ
We are ready for the soccer game
the Netherlands won the semi-final!