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Team:TU Delft/30 July 2010 content
From 2010.igem.org
Contents |
Lab work
Alkane Degradation
PCR Amplification
Yesterday's were ligation mixes were amplified with the primers PCR F and PCR R. The product was put on 1% agarose gel:
Lane Description:
| # | Description | Expected Length (bp) | Primers | Status | Remarks |
| 1 | PCR product of 007 + 008 | PCR F + PCR R | ✓ | ||
| 2 | PCR product of 009 + 010 | PCR F + PCR R | ✓ | ||
| 3 | PCR product of 017 + 018 | PCR F + PCR R | ✗ | ||
| 4 | PCR product of 019 + B0015 | PCR F + PCR R | ✓ | ||
| 5 | PCR product of 007 (negative control) | PCR F + PCR R | ✗ | The primers also anneal without a complete ligation | |
| M1 | SmartLadder | n/a | n/a | n/a |
Digestion
The PCR products were subsequently digested:
| # | Sample | Enzyme 1 | Enzyme 2 | Buffer | BSA | Needed fragment |
| 1 | 40 μL PCR product 007 + 008 | EcoRI | PstI | 2 (BioLabs) | ✓ | ‘E–007 + 008–P’ |
| 2 | 40 μL PCR product 009 + 010 | EcoRI | PstI | 2 (BioLabs) | ✓ | ‘E–009 + 010-P’ |
| 3 | 40 μL PCR product 017 + 018 | EcoRI | PstI | 2 (BioLabs) | ✓ | ‘E–017 + 018–P’ |
| 4 | 40 μL PCR product 019 + B0015 | EcoRI | PstI | 2 (BioLabs) | ✓ | ‘E–019 + B0015-P’ |
Alkane Sensing, Solvent Tolerance and Salt Tolerance
by Pieter
I tried to assemble the remaining biobricks from various sub projects. The goal is to assemble the following:
- AlkS + E0240
- PalkB + J06702
- J61101 + bbc1
- J61101 + PhPFDalpha
- J61101 + PhPFDbeta
To achieve this J06702, bbc1, PhPFDalpha and PhPFDbeta were amplified by PCR. The J61101 was cut open with S and P after which the biobricks were inserted. AlkS was inserted in the backbone with E0240.
PCR
The PCR protocol was followed for the amplification of the BioBricks.
PCR reactions
| # | Description | Expected lenght (bp) | Primer | Status |
| 1 | Marker | n/a | n/a | n/a |
| 2 | J06702 | 869 | G00100 + G00101 | ✗ |
| 3 | bbc1 | 703 | MF + MR2 | ✓ |
| 4 | PhPFDalpha | 528 | MF + MR2 | ✓ |
| 5 | PhPFDbeta | 430 | MF + MR2 | ✓ |
Digestion
The PCR products were subsequently digested:
| # | Sample | Enzyme 1 | Enzyme 2 | Buffer | BSA | Needed fragment |
| 1 | 2 μL AlkS | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–AlkS–S’ |
| 2 | 3 μL E0240 | EcoRI | XbaI | 2 (BioLabs) | ✓ | ‘X–E0240–E’ |
| 3 | 16 μL PalkB | SpeI | PstI | 2 (BioLabs) | ✓ | ‘P–PalkB–S’ |
| 4 | 40 μL J06702 PCR product | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–J06702–P’ |
| 5 | 6 μL J61101 | SpeI | PstI | 2 (BioLabs) | ✓ | ‘P–J61101–S’ |
| 6 | 40 μL bbc1 PCR product | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–J61101–P’ |
| 7 | 40 μL PhPFDalpha PCR product | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–PhPFDalpha–P’ |
| 8 | 40 μL PhPFDbeta PCR product | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–PhPFDalpha–P’ |
Ligation
The digestion products were ligated over two nights:
| # | BioBrick | Fragment 1 | Fragment 2 | Final volume |
| 1 | AlkS + E0240 | 10 μL ‘E–AlkS–S’ | 10 μL ‘X–E0240–E’ | 25 μL |
| 2 | J61101 + bbc1 | 7 μL ‘P–J61101–S’ | 1 μL ‘X–J61101–P’ | 10 μL |
| 3 | J61101 + PhPFDalpha | 7 μL ‘P–J61101–S’ | 1 μL ‘X–PhPFDalpha–P’ | 10 μL |
| 4 | J61101 + PhPFDbeta | 7 μL ‘P–J61101–S’ | 1 μL ‘X–PhPFDbeta–P’ | 10 μL |
