Team:TU Delft/27 July 2010 content

From 2010.igem.org

(Difference between revisions)
(→Alkane degradation)
 
(13 intermediate revisions not shown)
Line 1: Line 1:
 +
=Lab work=
 +
 +
==Hydrocarbon Sensing==
 +
 +
[https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=26_July_2010 Yesterday's] digestion products were checked on a gel.
 +
 +
[[Image:Geknipte ligatie 20100727.jpg|200px|thumb|left|1% agarose of plasmid check using digestion reaction. Gel runned at 100V for 1 hour. Of all samples 10 μL was loaded with 2 μL loadingbuffer. 5 μL was loaded of marker]]
 +
 +
Lane description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Restriction Enzymes'''
 +
|'''Expected Length (bp)'''
 +
|'''Status'''
 +
|-
 +
|1
 +
|marker
 +
|n/a
 +
|n/a
 +
|n/a
 +
|-
 +
|2
 +
|Undigested B0015
 +
|n/a
 +
|3318
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|3
 +
|Digested B0015
 +
|EcoRI, XbaI
 +
|15, 3303
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|4
 +
|Undigested E0422
 +
|n/a
 +
|2996
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|5
 +
|Digested E0422
 +
|EcoRI, XbaI
 +
|15, 2981
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|6
 +
|Undigested AlkS
 +
|n/a
 +
|4722
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|7
 +
|Digested AlkS
 +
|EcoRI, XbaI, NcoI
 +
|2673, 749, 1300
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|8
 +
|Digested AlkS
 +
|EcoRI, XbaI
 +
|2673, 2049
 +
|<font color=limegreen>✓</font>
 +
|}
 +
 +
All of the digestions turned out to be good! Let's ligate!
 +
 +
==Alkane degradation==
==Alkane degradation==
-
[https://2010.igem.org/Team:TU_Delft#/blog?blog=26_July_2010 Yesterday's] digestion products were checked on a gel.
+
[https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=26_July_2010 Yesterday's] digestion products were checked on a gel.
-
[[Image:TUDelft_Kira_digestion_2010_07_27.png|500px|thumb|left|Digestion]]
+
[[Image:TUDelft_Kira_digestion_2010_07_27.png|500px|thumb|left|1% agarose of plasmid check using digestion reaction. Gel runned at 100V for 1 hour. Of all samples 10 μL was loaded with 2 μL loadingbuffer. 5 μL was loaded of marker]]
 +
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"
|'''#'''
|'''#'''
-
|'''Lane description'''
+
|'''Description'''
-
|'''As expected?'''
+
|'''Expected length (bp)'''
 +
|'''Status'''
|-
|-
|1
|1
-
|[https://2010.igem.org/Image:TU_Delft_SmartLadder.jpg SmartLadder] (5μl)
+
|[https://2010.igem.org/Image:TU_Delft_SmartLadder.jpg SmartLadder]
-
|
+
|n/a
 +
|n/a
|-
|-
|2
|2
-
|rubA3 (cut)
+
|rubA3 + XbaI + PstI + PvuI
-
|✓
+
|198,
 +
|<font color=limegreen>✓</font>
|-
|-
|3
|3
-
|rubA3 (uncut)
+
|undigested rubA3
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|4
|4
-
|rubA4 (cut)
+
|rubA4 + XbaI + PstI + PvuI
-
|✓
+
|207,
 +
|<font color=limegreen>✓</font>
|-
|-
|5
|5
-
|rubA4 (uncut)
+
|undigested rubA4  
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|6
|6
-
|rubR (cut)
+
|rubR + XbaI + PstI + EcoRI
-
|✓
+
|1230
 +
|<font color=limegreen>✓</font>
|-
|-
|7
|7
-
|rubR (uncut)
+
|undigested rubR
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|8
|8
-
|ladA (cut)
+
|ladA + XbaI + PstI + PvuI
-
|✓
+
|1350,
 +
|<font color=limegreen>✓</font>
|-
|-
|9
|9
-
|ladA (uncut)
+
|undigested ladA  
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|10
|10
-
|ADH (cut)
+
|ADH + XbaI + PstI + EcoRI
-
|✓
+
|777,
 +
|<font color=limegreen>✓</font>
|-
|-
|11
|11
-
|ADH (uncut)
+
|undigested ADH
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|12
|12
-
|ALDH (cut)
+
|ALDH + XbaI + PstI + PvuI
-
|✓
+
|1521
 +
|<font color=limegreen>✓</font>
|-
|-
|13
|13
-
|ALDH (uncut)
+
|undigested ALDH  
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|14
|14
-
|J61100 (cut)
+
|J61100 + SpeI + PstI
-
|✓
+
|2116
 +
|<font color=limegreen>✓</font>
|-
|-
|15
|15
-
|J61100 (uncut)
+
|undigested J61100  
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|16
|16
-
|J61101 (cut)
+
|J61101 + SpeI + PstI
-
|✓
+
|2116
 +
|<font color=limegreen>✓</font>
|-
|-
|17
|17
-
|J61101 (uncut)
+
|undigested J61101  
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|-
|-
|18
|18
-
|J61107 (cut)
+
|J61107 + SpeI + PstI
-
|✓
+
|2116
 +
|<font color=limegreen>✓</font>
|-
|-
|19
|19
-
|J61107 (uncut)
+
|undigested J61107
-
|✓
+
|
 +
|<font color=limegreen>✓</font>
|}
|}
 +
As can be seen from the gel, all digestions went well, let's hope that the ligation / transformation also go well!
 +
====Transformation====
-
 
+
The [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=26_July_2010 ligation mixes] were incubated overnight. We [[Team:TU_Delft/protocols/transformation| transformed]] 10 μL of these ligations in Top10 competent cells, and these were grown on LB-plates with antibiotic over night.
-
<html><br/></html>
+
==RBS Characterization==
==RBS Characterization==
Line 94: Line 184:
[[Image:26_07_2010_Rbs.png]]
[[Image:26_07_2010_Rbs.png]]
-
Strength was calculated by taking the mean of the ratio between the expression of known RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and expression Anderson RBS over some time. Expression being the measured fluorescence divided by measured biomass (absorbance, OD).
+
Strength was calculated by taking the mean of the ratio between the expression of known RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and expression of Anderson RBS over some time. Expression being the measured fluorescence divided by measured biomass (absorbance, OD).
The measurements where taken from the part of the curve where optimal growth can be assumed, so from 0:40 until 2:30
The measurements where taken from the part of the curve where optimal growth can be assumed, so from 0:40 until 2:30
-
{|style="color:green; background-color:#ffffcc;" border="1" cellpadding="5"
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-
|RBS
+
|'''RBS'''
-
|Strength
+
|'''Strength'''
|-
|-
|[http://partsregistry.org/Part:BBa_J61100 J61100]
|[http://partsregistry.org/Part:BBa_J61100 J61100]

Latest revision as of 16:06, 15 August 2010

Contents

Lab work

Hydrocarbon Sensing

Yesterday's digestion products were checked on a gel.

1% agarose of plasmid check using digestion reaction. Gel runned at 100V for 1 hour. Of all samples 10 μL was loaded with 2 μL loadingbuffer. 5 μL was loaded of marker

Lane description:

# Description Restriction Enzymes Expected Length (bp) Status
1 marker n/a n/a n/a
2 Undigested B0015 n/a 3318 ✓
3 Digested B0015 EcoRI, XbaI 15, 3303 ✓
4 Undigested E0422 n/a 2996 ✓
5 Digested E0422 EcoRI, XbaI 15, 2981 ✓
6 Undigested AlkS n/a 4722 ✓
7 Digested AlkS EcoRI, XbaI, NcoI 2673, 749, 1300 ✓
8 Digested AlkS EcoRI, XbaI 2673, 2049 ✓

All of the digestions turned out to be good! Let's ligate!


Alkane degradation

Yesterday's digestion products were checked on a gel.

1% agarose of plasmid check using digestion reaction. Gel runned at 100V for 1 hour. Of all samples 10 μL was loaded with 2 μL loadingbuffer. 5 μL was loaded of marker

Lane description:

# Description Expected length (bp) Status
1 SmartLadder n/a n/a
2 rubA3 + XbaI + PstI + PvuI 198, ✓
3 undigested rubA3 ✓
4 rubA4 + XbaI + PstI + PvuI 207, ✓
5 undigested rubA4 ✓
6 rubR + XbaI + PstI + EcoRI 1230 ✓
7 undigested rubR ✓
8 ladA + XbaI + PstI + PvuI 1350, ✓
9 undigested ladA ✓
10 ADH + XbaI + PstI + EcoRI 777, ✓
11 undigested ADH ✓
12 ALDH + XbaI + PstI + PvuI 1521 ✓
13 undigested ALDH ✓
14 J61100 + SpeI + PstI 2116 ✓
15 undigested J61100 ✓
16 J61101 + SpeI + PstI 2116 ✓
17 undigested J61101 ✓
18 J61107 + SpeI + PstI 2116 ✓
19 undigested J61107 ✓

As can be seen from the gel, all digestions went well, let's hope that the ligation / transformation also go well!

Transformation

The ligation mixes were incubated overnight. We transformed 10 μL of these ligations in Top10 competent cells, and these were grown on LB-plates with antibiotic over night.

RBS Characterization

For our RBS characterization project, 5 different RBS sequences from the [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson Anderson RBS family] where placed in front of GFP and measured over 18 hours using a Gen5 fluorenscence and absorbance plate reader 26 07 2010 Rbs.png

Strength was calculated by taking the mean of the ratio between the expression of known RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and expression of Anderson RBS over some time. Expression being the measured fluorescence divided by measured biomass (absorbance, OD).

The measurements where taken from the part of the curve where optimal growth can be assumed, so from 0:40 until 2:30

RBS Strength
[http://partsregistry.org/Part:BBa_J61100 J61100] 0.047513
[http://partsregistry.org/Part:BBa_J61101 J61101] 0.119831
[http://partsregistry.org/Part:BBa_J61107 J61107] 0.065454
[http://partsregistry.org/Part:BBa_J61117 J61117] 0.038518
[http://partsregistry.org/Part:BBa_J61127 J61127] 0.087334
[http://partsregistry.org/Part:BBa_B0032 B0032] 0.300000

Next step: We want to relate the RBS sequence to the strength, so it might be possible to say predict something about the other Anderson RBS sequences.

Retrieved from "http://2010.igem.org/Team:TU_Delft/27_July_2010_content"