Team:TU Delft/4 August 2010 content

From 2010.igem.org

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=Alkane Sensing, Solvent Tolerance, Salt Tolerance, Emulsifier=
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=Lab work=
 +
 
 +
==Alkane Sensing, Solvent Tolerance, Salt Tolerance, Emulsifier==
''by Pieter''
''by Pieter''
We found it remarkable that the only positive samples we found so far were from the series without preamplifying the insert by PCR (e.g. the AlkS). This is why we decided to retry the insertion of bbc1, PhPFDalpha and PhPFDbeta in J61101, but this time without PCRing the insert.
We found it remarkable that the only positive samples we found so far were from the series without preamplifying the insert by PCR (e.g. the AlkS). This is why we decided to retry the insertion of bbc1, PhPFDalpha and PhPFDbeta in J61101, but this time without PCRing the insert.
-
==Digestion==
+
====Digestion====
For the assembly of BBa_K398101, BBa_K398205, BBa_K398206, BBa_K398402 and BBa_K398403 a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion]] was done:
For the assembly of BBa_K398101, BBa_K398205, BBa_K398206, BBa_K398402 and BBa_K398403 a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion]] was done:
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|}
|}
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=Alkane degradation=
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====Ligation====
 +
The digestion products were [[Team:TU_Delft/protocols/ligation|ligated]] in the afternoon:
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''BioBrick'''
 +
|'''Fragment 1'''
 +
|'''Fragment 2'''
 +
|'''Final volume'''
 +
|-
 +
|1
 +
|J61101 + bbc1
 +
|6 μL ‘X – bbc1 - P’
 +
|6 μL ‘P – J61101 - S’
 +
|20 μL
 +
|-
 +
|2
 +
|J61101 + PhPFDalpha
 +
|6 μL ‘P – J61101 – S’
 +
|6 μL ‘X – PhPFDalpha – P’
 +
|20 μL
 +
|-
 +
|3
 +
|J61101 + PhPFDbeta
 +
|6 μL ‘P – J61101 – S’
 +
|10 μL ‘X – PhPFDbeta – P’
 +
|20 μL
 +
|-
 +
|4
 +
|R0011-B0032 (9) + OprG-B0015
 +
|10 μL ‘P – R0011 - B0032 – S’
 +
|4 μL ‘X – OprG - B0015 – P’
 +
|20 μL
 +
|-
 +
|5
 +
|R0011-B0032 (12) + OprG-B0015
 +
|10 μL ‘P – R0011 - B0032 – S’
 +
|4.3 μL ‘X – OprG - B0015 – P’
 +
|20 μL
 +
|-
 +
|6
 +
|R0011-B0032 (9) + AlnA-B0015
 +
|10 μL ‘P – R0011 - B0032 – S’
 +
|4.3 μL ‘X – AlnA - B0015 – P’
 +
|20 μL
 +
|-
 +
|7
 +
|R0011-B0032 (9) + AlnA-B0015
 +
|10 μL ‘P – R0011 - B0032 – S’
 +
|4.3 μL ‘X – AlnA - B0015 – P’
 +
|20 μL
 +
|}
 +
 
 +
====Transformation====
 +
10 μL of ligation mixes 1 - 3 were transformed Top10 competent cells, and these were grown on LB-plates with antibiotic Amp over night.
 +
 
 +
10 μL of ligation mixes 4 - 7 were transformed DH5alpha competent cells, and these were grown on LB-plates with antibiotic Tet over night.
 +
 
 +
==Alkane degradation==
Today we started with a PCR of a number colonies from [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=3_August_2010 yesterday's] transformants. These had all given colonies on the plates, so hopefully they also contain the BioBricks! Five colonies from each plate were taken for the colony PCR, and after the PCR the products were analyzed on two gels.
Today we started with a PCR of a number colonies from [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=3_August_2010 yesterday's] transformants. These had all given colonies on the plates, so hopefully they also contain the BioBricks! Five colonies from each plate were taken for the colony PCR, and after the PCR the products were analyzed on two gels.
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[[Image:TUDelft_20100804_PCR1.png|400px|thumb|left|PCR 1]]
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[[Image:TUDelft_20100804_PCR1.png|450px|thumb|left|PCR 1]]
'''Lane description'''  
'''Lane description'''  
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|}
|}
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[[Image:TUDelft_20100804_PCR2.png|400px|thumb|left|PCR 2]]
+
[[Image:TUDelft_20100804_PCR2.png|450px|thumb|left|PCR 2]]
-
'''Lane description'''
+
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"
|'''#'''
|'''#'''
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|n/a
|n/a
|n/a
|n/a
 +
|}
 +
 +
==Hydrocarbon sensing==
 +
 +
====PCR Amplification====
 +
 +
To assembly the small parts like the promoters to the RBS were performed an overlapping PCR. For the parts that have to be connected to each other, primers were designed with a overlapping overhang. Then the parts were [[Team:TU_Delft/protocols/PCR|amplified]] using primers with the overhang. The product was put on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]:
 +
 +
Lane description:
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Primers'''
 +
|'''Expected length (bp)'''
 +
|'''Staus'''
 +
|-
 +
|1
 +
|1 μL PalkS12
 +
|MF + 304R
 +
|
 +
|
 +
|-
 +
|2
 +
|1 μL B0032
 +
|304F + G00101
 +
|
 +
|
 +
|-
 +
|3
 +
|1 μL P(Caif)
 +
|MF + 327R
 +
|
 +
|
 +
|-
 +
|4
 +
|1 μL B0032
 +
|327F + G00101
 +
|
 +
|
|}
|}

Latest revision as of 09:20, 13 August 2010

Contents

Lab work

Alkane Sensing, Solvent Tolerance, Salt Tolerance, Emulsifier

by Pieter

We found it remarkable that the only positive samples we found so far were from the series without preamplifying the insert by PCR (e.g. the AlkS). This is why we decided to retry the insertion of bbc1, PhPFDalpha and PhPFDbeta in J61101, but this time without PCRing the insert.

Digestion

For the assembly of BBa_K398101, BBa_K398205, BBa_K398206, BBa_K398402 and BBa_K398403 a digestion was done:

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 1 μg bbc1 XbaI PstI ScaI 2 (Biolabs) ✓ ‘X – bbc1 - P’
2 1 μg PhPFDalpha XbaI PstI ScaI 2 (Biolabs) ✓ ‘X – PhPFDalpha - P’
3 1 μg PhPFDbeta XbaI PstI ScaI 2 (Biolabs) ✓ ‘X – PhPFDbeta - P’
4 1 μg J61101 SpeI PstI 2 (Biolabs) ✓ ‘S – J61101 - P’
5 1 μg OprG-B0015 XbaI PstI 2 (Biolabs) ✓ ‘X – OprG-B0015 - P’
6 1 μg AlnA-B0015 XbaI PstI 2 (Biolabs) ✓ ‘X – AlnA-B0015 - P’
7 1 μg R0011-B0032 (sample 9) SpeI PstI 2 (Biolabs) ✓ ‘S – R0011-B0032 - P’
8 1 μg R0011-B0032 (sample 10) SpeI PstI 2 (Biolabs) ✓ ‘S – R0011-B0032 - P’

Ligation

The digestion products were ligated in the afternoon:

# BioBrick Fragment 1 Fragment 2 Final volume
1 J61101 + bbc1 6 μL ‘X – bbc1 - P’ 6 μL ‘P – J61101 - S’ 20 μL
2 J61101 + PhPFDalpha 6 μL ‘P – J61101 – S’ 6 μL ‘X – PhPFDalpha – P’ 20 μL
3 J61101 + PhPFDbeta 6 μL ‘P – J61101 – S’ 10 μL ‘X – PhPFDbeta – P’ 20 μL
4 R0011-B0032 (9) + OprG-B0015 10 μL ‘P – R0011 - B0032 – S’ 4 μL ‘X – OprG - B0015 – P’ 20 μL
5 R0011-B0032 (12) + OprG-B0015 10 μL ‘P – R0011 - B0032 – S’ 4.3 μL ‘X – OprG - B0015 – P’ 20 μL
6 R0011-B0032 (9) + AlnA-B0015 10 μL ‘P – R0011 - B0032 – S’ 4.3 μL ‘X – AlnA - B0015 – P’ 20 μL
7 R0011-B0032 (9) + AlnA-B0015 10 μL ‘P – R0011 - B0032 – S’ 4.3 μL ‘X – AlnA - B0015 – P’ 20 μL

Transformation

10 μL of ligation mixes 1 - 3 were transformed Top10 competent cells, and these were grown on LB-plates with antibiotic Amp over night.

10 μL of ligation mixes 4 - 7 were transformed DH5alpha competent cells, and these were grown on LB-plates with antibiotic Tet over night.

Alkane degradation

Today we started with a PCR of a number colonies from yesterday's transformants. These had all given colonies on the plates, so hopefully they also contain the BioBricks! Five colonies from each plate were taken for the colony PCR, and after the PCR the products were analyzed on two gels.

Of BioBricks 007A, 012K, 021A and 021K there were a number of successful PCRs. Overnight an extra PCR will be done on some extra colonies from 020A. This was the only 3-way ligation done, which is probably why it's the only one that was unsucessful. The reason 011A failed was because the BrioBrick used to create this (007T) was wrong after all (seen during the digestion check of yesterday


PCR 1

Lane description

# Description Primers Expected length (bp) ✓ ✗
L SmartLadder n/a n/a n/a n/a
1-5 007A G00100 + G00101 1538 1,2,4 3,5
6-10 011A G00100 + G00101 1816 none 6,7,8,9,10
11-15 012K G00100 + G00101 1720 3,5 1,2,4
L SmartLadder n/a n/a n/a n/a
PCR 2

Lane description

# Description Primers Expected length (bp) ✓ ✗
L SmartLadder n/a n/a n/a n/a
1-5 020K G00100 + G00101 2428 none 1,2,3,4,5
6-10 021A G00100 + G00101 2011 8,10 6,7,9
11-15 021K G00100 + G00101 2011 11,12,13,15 14
16 021KA G00100 + G00101 2011 ✓
L SmartLadder n/a n/a n/a n/a

Hydrocarbon sensing

PCR Amplification

To assembly the small parts like the promoters to the RBS were performed an overlapping PCR. For the parts that have to be connected to each other, primers were designed with a overlapping overhang. Then the parts were amplified using primers with the overhang. The product was put on 1% agarose gel:

Lane description:

# Description Primers Expected length (bp) Staus
1 1 μL PalkS12 MF + 304R
2 1 μL B0032 304F + G00101
3 1 μL P(Caif) MF + 327R
4 1 μL B0032 327F + G00101
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