Team:TU Delft/27 July 2010 content

From 2010.igem.org

(Difference between revisions)
(→RBS Characterization)
Line 1: Line 1:
==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#/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'''
 +
|'''Remarks'''
|-
|-
|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
 +
|
|✓
|✓
 +
|
|-
|-
|3
|3
-
|rubA3 (uncut)
+
|undigested rubA3
 +
|
|✓
|✓
 +
|
|-
|-
|4
|4
-
|rubA4 (cut)
+
|rubA4 + XbaI + PstI + PvuI
 +
|
|✓
|✓
 +
|
|-
|-
|5
|5
-
|rubA4 (uncut)
+
|undigested rubA4  
 +
|
|✓
|✓
 +
|
|-
|-
|6
|6
-
|rubR (cut)
+
|rubR + XbaI + PstI + EcoRI
 +
|
|✓
|✓
 +
|
|-
|-
|7
|7
-
|rubR (uncut)
+
|undigested rubR  
 +
|
|✓
|✓
 +
|
|-
|-
|8
|8
-
|ladA (cut)
+
|ladA + XbaI + PstI + PvuI
 +
|
|✓
|✓
 +
|
|-
|-
|9
|9
-
|ladA (uncut)
+
|undigested ladA  
 +
|
|✓
|✓
 +
|
|-
|-
|10
|10
-
|ADH (cut)
+
|ADH + XbaI + PstI + EcoRI
 +
|
|✓
|✓
 +
|
|-
|-
|11
|11
-
|ADH (uncut)
+
|undigested ADH
 +
|
|✓
|✓
 +
|
|-
|-
|12
|12
-
|ALDH (cut)
+
|ALDH + XbaI + PstI + PvuI
 +
|
|✓
|✓
 +
|
|-
|-
|13
|13
-
|ALDH (uncut)
+
|undigested ALDH  
 +
|
|✓
|✓
 +
|
|-
|-
|14
|14
-
|J61100 (cut)
+
|J61100 + SpeI + PstI
 +
|
|✓
|✓
 +
|
|-
|-
|15
|15
-
|J61100 (uncut)
+
|undigested J61100  
 +
|
|✓
|✓
 +
|
|-
|-
|16
|16
-
|J61101 (cut)
+
|J61101 + SpeI + PstI
 +
|
|✓
|✓
 +
|
|-
|-
|17
|17
-
|J61101 (uncut)
+
|undigested J61101  
 +
|
|✓
|✓
 +
|
|-
|-
|18
|18
-
|J61107 (cut)
+
|J61107 + SpeI + PstI
 +
|
|✓
|✓
 +
|
|-
|-
|19
|19
-
|J61107 (uncut)
+
|undigested J61107
 +
|
|✓
|✓
 +
|
|}
|}
-
'''Transformation'''
+
<h4>Transformation</h4>
The [https://2010.igem.org/Team:TU_Delft#/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
The [https://2010.igem.org/Team:TU_Delft#/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
Line 96: Line 137:
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]

Revision as of 06:35, 2 August 2010

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 Remarks
1 SmartLadder n/a n/a
2 rubA3 + XbaI + PstI + PvuI ✓
3 undigested rubA3 ✓
4 rubA4 + XbaI + PstI + PvuI ✓
5 undigested rubA4 ✓
6 rubR + XbaI + PstI + EcoRI ✓
7 undigested rubR ✓
8 ladA + XbaI + PstI + PvuI ✓
9 undigested ladA ✓
10 ADH + XbaI + PstI + EcoRI ✓
11 undigested ADH ✓
12 ALDH + XbaI + PstI + PvuI ✓
13 undigested ALDH ✓
14 J61100 + SpeI + PstI ✓
15 undigested J61100 ✓
16 J61101 + SpeI + PstI ✓
17 undigested J61101 ✓
18 J61107 + SpeI + PstI ✓
19 undigested J61107 ✓

Transformation

The ligation mixes were incubated overnight. We transformed 10 μL of these ligations in Top10 competent cells

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"