Team:TU Delft/6 July 2010 content

From 2010.igem.org

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===Lab work===
+
=Lab work=
 +
==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!
-
We were so excited; we immediately solved the DNA in water and performed a [[Team:TU_Delft/protocols/transformation|transformation]] with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.  
+
We were so excited; we immediately dissolved the DNA in water and performed a [[Team:TU_Delft/protocols/transformation|transformation]] with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.  
-
1. AlkB2 on 100 μg/mL Ampicillin
+
* 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)
-
2. rubA3 on 100 μg/mL 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 [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]]:
-
3. rubA4 on 100 μg/mL Ampicillin
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''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’
 +
|}
-
4. rubB on 50 μg/mL Kanamycin
+
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:
-
5. ladA on 100 μg/mL Ampicillin
+
[[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]]
-
6. ADH on 100 μg/mL Ampicillin
+
Lane description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Status'''
 +
|-
 +
|M1
 +
|SmartLadder marker (5 μL)
 +
|n/a
 +
|n/a
 +
|-
 +
|1
 +
|Undigested J61100 in pSB1A2
 +
|2134
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|3
 +
|Undigested J61101 in pSB1A2
 +
|2134
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|4
 +
|J61100 + SpeI + PstI 
 +
|18, 2116
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|5
 +
|J61101 + SpeI + PstI 
 +
|18, 2116
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|6
 +
|Undigested J61107 in pSB1A2
 +
|2134
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|7
 +
|J61107 + SpeI + PstI
 +
|18, 2116
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|8
 +
|Undigested J61117 in pSB1A2
 +
|2134
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|9
 +
|J61117 + SpeI + PstI 
 +
|18, 2116
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|10
 +
|Undigested J61127 in pSB1A2
 +
|2134
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|11
 +
|J61127 + SpeI + PstI
 +
|18, 2116
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|12
 +
|Undigested I13401 in pSB1A2
 +
|2936
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|13
 +
|I13401 + XbaI + PstI
 +
|883, 2053
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|14
 +
|empty
 +
|empty
 +
|empty
 +
|-
 +
|15
 +
|BioRad EZ Load
 +
|n/a
 +
|n/a
 +
|}
-
7. ALDH on 50 μg/mL Kanamycin
+
Followed by over night [https://2010.igem.org/Team:TU_Delft/protocols/ligation ligation]:
-
8. bbc1 on 100 μg/mL Ampicillin
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-
 
+
|'''#'''
-
9. AlnA on 100 μg/mL Ampicillin
+
|'''BioBrick'''
-
 
+
|'''Recipient plasmid'''
-
10. OprG on 100 μg/mL Ampicillin
+
|'''Fragment'''
-
 
+
|'''Final volume'''
-
11. PalkS1-2 on 100 μg/mL Ampicillin
+
|-
-
 
+
|1
-
12. PalkB on 100 μg/mL Ampicillin
+
|K398500A
-
 
+
|130 μg ‘S–J61100 pSB1A2–P’
-
13. P(CaiF) on 100 μg/mL Ampenicillin
+
|154 μg ‘X-I13401-P’
-
 
+
|26 μL
-
14. AlkS on 50 μg/mL Kanamycin
+
|-
-
 
+
|2
-
15. PhPFD-α on 100 μg/mL Ampicillin
+
|K398501A
-
 
+
|206 μg ‘S–J61101 pSB1A2–P’
-
16. PhPFD-β on 100 μg/mL Ampicillin
+
|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:  
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:  
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-
|
+
|'''#'''
-
| Hexane (mL)
+
|'''Hexane (mL)'''
-
| Tris Buffer pH 8 (mL)
+
|'''Tris Buffer pH 8 (mL)'''
-
| 10% SDS (mL)
+
|'''10% SDS (mL)'''
|-
|-
|B
|B
Line 77: Line 268:
|}
|}
-
<b> Characterization of Anderson RBS sequences </b>
+
=Kampioenen!!!!=
-
 
+
-
Yesterday's purified PCR product of BBa_I13401 was [[digested]] using XbaI and PstI in order to obtain a back-insert. The relevant Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were digested using SpeI and PstI. Purification of the digestion products using Roche's PCR Purification Kit and subsequent overnight ligation yielded the 5 different RBS sequences now containing the GFP reporter regions.
+
-
 
+
-
===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!
Retrieved from "http://2010.igem.org/Team:TU_Delft/6_July_2010_content"