Team:TU Delft/2 August 2010 content

From 2010.igem.org

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-
=Alkane Sensing, Solvent Tolerance and Salt Tolerance=
+
=Lab work=
 +
==Alkane Sensing, Solvent Tolerance and Salt Tolerance==
''by Pieter''
''by Pieter''
The plates containing yesterday's ligations contained colonies, to check whether they really contain the desired BioBrick a [[Team:TU_Delft/protocols/colony PCR|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.
The plates containing yesterday's ligations contained colonies, to check whether they really contain the desired BioBrick a [[Team:TU_Delft/protocols/colony PCR|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.
-
=Alkane degradation=
+
[[Image:TU_Delft_Pi2_Colony_PCR_part_1.png|400px]]
 +
 
 +
* BBa_K398305 = Alks -> E0240
 +
* BBa_K398101 = bbc1 -> J61101
 +
* BBa_K398402 = PhPFDalpha -> J61101
 +
* BBa_K398403 = PhPFDbeta -> J61101
 +
 
 +
Lane Description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|'''Remarks'''
 +
|-
 +
|M
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|1
 +
|BBa_K398305
 +
|3772
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|2
 +
|BBa_K398305
 +
|3772
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|3
 +
|BBa_K398305
 +
|3772
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|4
 +
|BBa_K398305
 +
|3772
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|5
 +
|BBa_K398305
 +
|3772
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|6
 +
|BBa_K398101
 +
|1007
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|7
 +
|BBa_K398101
 +
|1007
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|8
 +
|BBa_K398101
 +
|1007
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|m
 +
|EZ Ladder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|M
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|1
 +
|BBa_K398101
 +
|1007
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|2
 +
|BBa_K398101
 +
|1007
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|3
 +
|BBa_K398402
 +
|836
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|4
 +
|BBa_K398402
 +
|836
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|5
 +
|BBa_K398402
 +
|836
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|6
 +
|BBa_K398402
 +
|836
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|7
 +
|BBa_K398402
 +
|836
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|8
 +
|BBa_K398403
 +
|734
 +
|G00101 + G00100
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|m
 +
|EZ Ladder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|}
 +
 
 +
[[Image:TU_Delft_Pi2_Colony_PCR_part_2.png|400px]]
 +
 
 +
Lane Description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''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
 +
|<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
 +
|<font color=red>X</font>
 +
|
 +
|-
 +
|M
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|}
 +
 
 +
==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 [[Team:TU_Delft/protocols/colony PCR|colony PCR]] on all 12 white colonies.
 +
 
 +
[[Image:TU_Delft_Pi3_Colony_PCR.jpg|400px]]
 +
 
 +
*BBa_K398202 = R0011 + B0032
 +
 
 +
Lane Description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|'''Remarks'''
 +
|-
 +
|M
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|1
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|2
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|3
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|4
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|5
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|6
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|7
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|8
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|9
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|Isolated plasmid
 +
|-
 +
|10
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|11
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|12
 +
|BBa_K398202
 +
|392
 +
|G00101 + G00100
 +
|
 +
|Isolated plasmid
 +
|}
 +
 
 +
None of the bands is truly positive. Still number 4,5 and 9 could be positive. Or due to the GC content the bands could be shifted a little bit upwards, so that maybe even sample 12 could be positive.
 +
 
 +
Just to be sure, we isolated the plasmids and send them to BaseClear for sequencing. The results will be in by Thursday.
 +
 
 +
==Alkane degradation==
For the next step in BioBrick formation a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion]] was done:
For the next step in BioBrick formation a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion]] was done:
Line 92: Line 430:
Results of the digestion on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]   
Results of the digestion on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]   
-
[[Image:TUDelft_20100802_digestion.png|400px|1% Agarose of digestion check ]]
+
[[Image:TUDelft_20100802_digestion.png|400px|thumb|left|1% Agarose of digestion check ]]
-
gel runned at 100V for 1 hour
+
The gel was runned at 100V for 1 hour, loaded 5 μL of marker and 5 μL sample + 1 μL loadingbuffer
-
loaded 5 μL of marker and 10 μL sample + 2 μL loadingbuffer
+
Lane description:
Lane description:
Line 102: Line 439:
|'''Expected Length (bp)'''
|'''Expected Length (bp)'''
|'''Status'''
|'''Status'''
-
|''Remarks'''
 
-
|-
 
-
|
 
-
|marker
 
-
|
 
|-
|-
|1
|1
-
|Undigested part name + vector (e.g. B0015 in pSB1AK3)
+
|Smartladder
|
|
-
|✓or ✗
 
|
|
|-
|-
|2
|2
-
|Part name + vector (e.g. B0015 in pSB1AK3) + Enzyme 1 + Enzyme 2
+
|‘E – J61100-AlkB2 – S’ (007T)
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=red>✗</font>
|-
|-
|3
|3
-
|Undigested part
+
|Undigested 007T
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=red>✗</font>
|-
|-
|4
|4
-
|Part + Enzyme 1 + Enzyme 2
+
|‘E – J61100-rubA3 – X’ (008A)
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|5
|5
-
|Undigested part
+
|Undigested 008A
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|6
|6
-
|Part + Enzyme 1 + Enzyme 2
+
|‘E – J61100-rubR – S’ (010A)
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|7
|7
-
|Undigested part
+
|Undigested 010A
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|8
|8
-
|Part + Enzyme 1 + Enzyme 2
+
|‘E – B0015 – pSB1AK3 – X’
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|9
|9
-
|Undigested part
+
|pSB1AK3-B0015
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|10
|10
-
|Part + Enzyme 1 + Enzyme 2
+
|‘E – J61100-ladA – S’ (017A)
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|11
|11
-
|Undigested part
+
|Undigested 017A
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|12
|12
-
|Part + Enzyme 1 + Enzyme 2
+
|‘X – J61101-ADH – P’ (018A)
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|13
|13
-
|Undigested part
+
|Undigested 018A
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|14
|14
-
|Part + Enzyme 1 + Enzyme 2
+
|‘E – J61107-ALDH – S’ (019A)
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|15
|15
-
|Undigested part
+
|Undigested (019A)
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|16
|16
-
|Part + Enzyme 1 + Enzyme 2
+
|‘E – pSB1K3 – P’
-
|
+
-
|✓or ✗
+
|
|
 +
|<font color=limegreen>✓</font>
|-
|-
|17
|17
-
|Undigested part
+
|Smartladder
-
|
+
-
|✓or ✗
+
-
|
+
-
|-
+
-
|18
+
-
|Part + Enzyme 1 + Enzyme 2
+
-
|
+
-
|✓or ✗
+
-
|
+
-
|-
+
-
|19
+
-
|Undigested part
+
-
|
+
-
|✓or ✗
+
-
|
+
-
|-
+
-
|20
+
-
|Part + Enzyme 1 + Enzyme 2
+
|
|
-
|✓or ✗
 
|
|
|}
|}

Latest revision as of 16:08, 15 August 2010

Contents

Lab work

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
M SmartLadder n/a n/a n/a

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.

TU Delft Pi3 Colony PCR.jpg

  • BBa_K398202 = R0011 + B0032

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1 BBa_K398202 392 G00101 + G00100
2 BBa_K398202 392 G00101 + G00100
3 BBa_K398202 392 G00101 + G00100
4 BBa_K398202 392 G00101 + G00100
5 BBa_K398202 392 G00101 + G00100
6 BBa_K398202 392 G00101 + G00100
7 BBa_K398202 392 G00101 + G00100
8 BBa_K398202 392 G00101 + G00100
9 BBa_K398202 392 G00101 + G00100 Isolated plasmid
10 BBa_K398202 392 G00101 + G00100
11 BBa_K398202 392 G00101 + G00100
12 BBa_K398202 392 G00101 + G00100 Isolated plasmid

None of the bands is truly positive. Still number 4,5 and 9 could be positive. Or due to the GC content the bands could be shifted a little bit upwards, so that maybe even sample 12 could be positive.

Just to be sure, we isolated the plasmids and send them to BaseClear for sequencing. The results will be in by Thursday.

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"