Team:TU Delft/2 August 2010 content

From 2010.igem.org

(Difference between revisions)
(→Alkane Sensing, Solvent Tolerance and Salt Tolerance)
(→Alkane Sensing, Solvent Tolerance and Salt Tolerance)
Line 174: Line 174:
|M
|M
|SmartLadder
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|1 - 7
 +
|Eva's samples
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|m
 +
|EZ tLadder
|n/a
|n/a
|n/a
|n/a
Line 180: Line 194:
|-
|-
|1
|1
-
|BBa_K398305
+
|BBa_K398403
-
|3772
+
|734
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|2
 +
|BBa_K398403
 +
|734
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|3
 +
|BBa_K398403
 +
|734
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|4
 +
|BBa_K398403
 +
|734
|G00101 + G00100
|G00101 + G00100
|<font color=red>X</font>
|<font color=red>X</font>

Revision as of 09:11, 4 August 2010

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

The plates containing yesterday's ligations contained colonies, to check whether they really contain the desired BioBrick a colony PCR was done, and the used colonies were grown in liquid LB medium over night. The results from the PCR were analysed on a 1% agarose gel.

TU Delft Pi2 Colony PCR part 1.png

  • BBa_K398305 = Alks -> E0240
  • BBa_K398101 = bbc1 -> J61101
  • BBa_K398402 = PhPFDalpha -> J61101
  • BBa_K398403 = PhPFDbeta -> J61101

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1 BBa_K398305 3772 G00101 + G00100 X
2 BBa_K398305 3772 G00101 + G00100 X
3 BBa_K398305 3772 G00101 + G00100 X
4 BBa_K398305 3772 G00101 + G00100 X
5 BBa_K398305 3772 G00101 + G00100 X
6 BBa_K398101 1007 G00101 + G00100 X
7 BBa_K398101 1007 G00101 + G00100 X
8 BBa_K398101 1007 G00101 + G00100 X
m EZ Ladder n/a n/a n/a
M SmartLadder n/a n/a n/a
1 BBa_K398101 1007 G00101 + G00100 X
2 BBa_K398101 1007 G00101 + G00100 X
3 BBa_K398402 836 G00101 + G00100 X
4 BBa_K398402 836 G00101 + G00100 X
5 BBa_K398402 836 G00101 + G00100 X
6 BBa_K398402 836 G00101 + G00100 X
7 BBa_K398402 836 G00101 + G00100 X
8 BBa_K398403 734 G00101 + G00100 X
m EZ Ladder n/a n/a n/a

TU Delft Pi2 Colony PCR part 2.png

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1 - 7 Eva's samples n/a n/a n/a
m EZ tLadder n/a n/a n/a
1 BBa_K398403 734 G00101 + G00100 X
2 BBa_K398403 734 G00101 + G00100 X
3 BBa_K398403 734 G00101 + G00100 X
4 BBa_K398403 734 G00101 + G00100 X

Emulsifier

When going over the plates in the fridge we noticed a few white colonies on plates from 13-07-'10. Previously we thought there were only red colonies. So we decided to do a colony PCR on all 12 white colonies.

Alkane degradation

For the next step in BioBrick formation a digestion was done:

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 1 μg 007T EcoRI SpeI BamH1 2 (Biolabs) ✓ ‘E – J61100-AlkB2 – S’
2 1 μg 008A EcoRI XbaI 2 (Biolabs) ✓ ‘E – J61100-rubA3 – X’
3 1 μg 010A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E – J61100-rubR – S’
4 2 μg B0015 EcoRI XbaI 2 (Biolabs) ✓ ‘E – B0015 – pSB1AK3 – X’
5 1 μg 017A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E – J61100-ladA – S’
6 1 μg 018A XbaI PstI AseI 2 (Biolabs) ✓ ‘X – J61101-ADH – P’
7 1 μg 019A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E – J61107-ALDH – S’
8 1 μg pSB1K3 EcoRI PstI 2 (Biolabs) ✓ ‘E – pSB1K3 – P’

Results of the digestion on 1% agarose gel

1% Agarose of digestion check

The gel was runned at 100V for 1 hour, loaded 5 μL of marker and 5 μL sample + 1 μL loadingbuffer

Lane description:

# Description Expected Length (bp) Status
1 Smartladder
2 ‘E – J61100-AlkB2 – S’ (007T) ✗
3 Undigested 007T ✗
4 ‘E – J61100-rubA3 – X’ (008A) ✓
5 Undigested 008A ✓
6 ‘E – J61100-rubR – S’ (010A) ✓
7 Undigested 010A ✓
8 ‘E – B0015 – pSB1AK3 – X’ ✓
9 pSB1AK3-B0015 ✓
10 ‘E – J61100-ladA – S’ (017A) ✓
11 Undigested 017A ✓
12 ‘X – J61101-ADH – P’ (018A) ✓
13 Undigested 018A ✓
14 ‘E – J61107-ALDH – S’ (019A) ✓
15 Undigested (019A) ✓
16 ‘E – pSB1K3 – P’ ✓
17 Smartladder
Retrieved from "http://2010.igem.org/Team:TU_Delft/2_August_2010_content"