Team:TU Delft/6 July 2010 content

From 2010.igem.org

(Difference between revisions)
(→Lab work)
 
(17 intermediate revisions not shown)
Line 1: Line 1:
-
===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:
|}
|}
-
 
+
=Kampioenen!!!!=
-
<b> Characterization of Anderson RBS sequences </b>
+
-
 
+
-
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 [https://2010.igem.org/Team:TU_Delft/protocols/ligation 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"