Team:TU Delft/4 August 2010 content
From 2010.igem.org
(8 intermediate revisions not shown) | |||
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- | =Alkane Sensing, Solvent Tolerance, Salt Tolerance, Emulsifier= | + | =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: | ||
Line 90: | Line 92: | ||
|} | |} | ||
- | = | + | ====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. | ||
+ | |||
+ | 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 [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=3_August_2010 yesterday] | ||
+ | |||
+ | |||
+ | [[Image:TUDelft_20100804_PCR1.png|450px|thumb|left|PCR 1]] | ||
- | 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" | ||
|'''#''' | |'''#''' | ||
Line 121: | Line 184: | ||
|011A | |011A | ||
|G00100 + G00101 | |G00100 + G00101 | ||
- | | | + | |1816 |
|none | |none | ||
|6,7,8,9,10 | |6,7,8,9,10 | ||
Line 128: | Line 191: | ||
|012K | |012K | ||
|G00100 + G00101 | |G00100 + G00101 | ||
- | | | + | |1720 |
|3,5 | |3,5 | ||
|1,2,4 | |1,2,4 | ||
Line 140: | Line 203: | ||
|} | |} | ||
- | [[Image:TUDelft_20100804_PCR2.png| | + | [[Image:TUDelft_20100804_PCR2.png|450px|thumb|left|PCR 2]] |
+ | |||
+ | Lane description | ||
+ | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | ||
+ | |'''#''' | ||
+ | |'''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 [[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
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 |
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 |