Team:TU Delft/3 August 2010 content

From 2010.igem.org

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=Alkane degradation=
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=Lab work=
-
[https://2010.igem.org/Team:TU_Delft#/blog?blog=2_August_2010 Yesterday's] digestions showed that BioBrick 007 was not the expected length. From this it was concluded that the plasmid thus does not contain the desired insert, so we will make the BioBrick again. This was done, starting with a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion]]:
+
==Alkane Sensing, Solvent Tolerance and Salt Tolerance==
 +
''by Pieter''
 +
 
 +
Because yesterday's Colony PCRs all turned out negative, we decided to pick an additional 8 colonies per plate and perform [https://2010.igem.org/Team:TU_Delft/protocols/colony_PCR Colony PCR] on them.
 +
 
 +
[[Image:TU_Delft_Pi2_Colony_PCR_retry_part_1.png|400px]]
 +
 
 +
* BBa_K398305 = Alks -> E0240
 +
* BBa_K398101 = bbc1 -> J61101
 +
* BBa_K398402 = PhPFDalpha -> J61101
 +
 
 +
Lane Description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|'''Remarks'''
 +
|-
 +
|1-8
 +
|BBa_K398305
 +
|3772
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|M
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|9-16
 +
|BBa_K398101
 +
|1007
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|M
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|-
 +
|17-24
 +
|BBa_K398402
 +
|836
 +
|G00101 + G00100
 +
|
 +
|
 +
|}
 +
 
 +
[[Image:TU_Delft_Pi2_Colony_PCR_retry_part_2.png|400px]]
 +
 
 +
* 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
 +
|
 +
|-
 +
|1-8
 +
|BBa_K398403
 +
|734
 +
|G00101 + G00100
 +
|
 +
|
 +
|-
 +
|M
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|}
 +
 
 +
BBa_K398305 sample 6 and 8 were considered the only positive colonies. Tomorrow we will isolate the plasmids and prepare -80 stocks.
 +
 
 +
==Alkane degradation==
 +
 
 +
[https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=2_August_2010 Yesterday's] digestions showed that BioBrick 007 was not the expected length. From this it was concluded that the plasmid thus does not contain the desired insert, so we will make the BioBrick again. This was done, starting with a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion]]. An extra cut was done in the backbone of alkB2 to decrease the chances of religation.
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
Line 20: Line 115:
|2 (Biolabs)
|2 (Biolabs)
|✓
|✓
-
|‘S – J61100-pSB1A2 – P’
+
|‘S – pSB1A2 - J61100– P’
|-
|-
|2
|2
Line 33: Line 128:
|}
|}
-
The digestions were checked on a gel:
+
The digestions were done during 2 hours, and checked on a gel:
 +
 
 +
[[Image:TUDelft_20100803_digestion.png|300px|thumb|left|Digestion check]]
Lane description
Lane description
Line 48: Line 145:
|-
|-
|1
|1
-
|‘S – J61100-pSB1A2 – P’
+
|‘S – pSB1A2 - J61100 – P’
|2116
|2116
-
|
+
|✓
|-
|-
|2
|2
-
|undigested J61100-pSB1A2
+
|undigested pSB1A2-J61100
-
|
+
|
|
 +
|✓
|-
|-
|3
|3
|‘X – alkB2 – P’
|‘X – alkB2 – P’
|1263
|1263
-
|
+
|✓
|-
|-
|4
|4
|Undigested alkB2
|Undigested alkB2
|
|
-
|
+
|✓
|-
|-
|}
|}
 +
 +
Directly following the digestion, the products were [[Team:TU_Delft/protocols/ligation|ligated]] for 4 hours:
 +
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''BioBrick'''
 +
|'''Fragment 1'''
 +
|'''Fragment 2'''
 +
|-
 +
|1
 +
|pSB1A2-J61100-alkB2
 +
|‘X – alkB2 – P’
 +
|‘S – pSB1A2-J61100 – P’
 +
|-
 +
|}
 +
 +
10 μL of this ligation mix aswell as [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=2_August_2010 yesterday's] ligations were used to [[Team:TU_Delft/protocols/transformation| transform]] Top10 competent cells.

Latest revision as of 08:01, 7 August 2010

Lab work

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

Because yesterday's Colony PCRs all turned out negative, we decided to pick an additional 8 colonies per plate and perform Colony PCR on them.

TU Delft Pi2 Colony PCR retry part 1.png

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

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
1-8 BBa_K398305 3772 G00101 + G00100
M SmartLadder n/a n/a n/a
9-16 BBa_K398101 1007 G00101 + G00100
M SmartLadder n/a n/a n/a
17-24 BBa_K398402 836 G00101 + G00100

TU Delft Pi2 Colony PCR retry part 2.png

  • BBa_K398403 = PhPFDbeta -> J61101

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1-8 BBa_K398403 734 G00101 + G00100
M SmartLadder n/a n/a n/a

BBa_K398305 sample 6 and 8 were considered the only positive colonies. Tomorrow we will isolate the plasmids and prepare -80 stocks.

Alkane degradation

Yesterday's digestions showed that BioBrick 007 was not the expected length. From this it was concluded that the plasmid thus does not contain the desired insert, so we will make the BioBrick again. This was done, starting with a digestion. An extra cut was done in the backbone of alkB2 to decrease the chances of religation.

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 J61100 SpeI PstI ✗ 2 (Biolabs) ✓ ‘S – pSB1A2 - J61100– P’
2 1 μg alkB2 XbaI PstI AseI 2 (Biolabs) ✓ ‘X – alkB2 – P’

The digestions were done during 2 hours, and checked on a gel:

Digestion check

Lane description

# Description Expected Length (bp) Status
0 Smartladder (5μl)
1 ‘S – pSB1A2 - J61100 – P’ 2116 ✓
2 undigested pSB1A2-J61100 ✓
3 ‘X – alkB2 – P’ 1263 ✓
4 Undigested alkB2 ✓

Directly following the digestion, the products were ligated for 4 hours:

# BioBrick Fragment 1 Fragment 2
1 pSB1A2-J61100-alkB2 ‘X – alkB2 – P’ ‘S – pSB1A2-J61100 – P’

10 μL of this ligation mix aswell as yesterday's ligations were used to transform Top10 competent cells.

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