Team:TU Delft/30 July 2010 content

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Contents

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 with PFX

Per sample the following mix was used:

  • 1,5 ul 10 mM dNTPs
  • 5 ul Enhancer
  • 5 ul 10x PFX Buffer
  • 1 ul 50 mM MgSO4
  • 3 ul 5 pmol/ul Forward Primer
  • 3 ul 5 pmol/ul Reverse Primer
  • 0.6 ul PFX Polymerase
  • 1 ul DNA Template
  • 29.9 ul H2O

PCR Program: start:

  • 2 min 95 C
  • 1 min 50 C
  • 1 min 68 C

repeated 25 times:

  • 1 min 95 C
  • 1 min 50 C
  • 1 min 68 C

end:

  • 10 min 68 C

PCR reactions

# Description Expected lenght (bp) Primer Status Remarks
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
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