User:Andrew Perry/Notebook/bbGibson/2011/06/10

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Single part Gibson assembly

This is my first attempt at a simple vector + single part Gibson assembly reaction.

Set up assembly reactions on ice:

  • 15 ul Gibson master mix
  • 1 uL vector (V.f/V.r) (~50 ng total)
  • 1 uL part (I13522 A.f/END.r) (~50 ng total)
  • 3 uL water

20 ul total volume.

  • Incubated 50 °C, 60 mins.
  • Transformed into 50 ul DH5α competent cells
  • (1 hour on ice, heat shock 1 min 42 °C, + 200 uL SOC, 37 °C shaking 1 hour, plated on LB chlor or LB tet agar plates)
  • Grew plates O/N 37 °C, then left at 4 °C.


Did reactions:

  • linear pSB1C3 V.f/V.r only
  • linear pSB1T3 V.f/V.r only
  • linear pSB1C3 V.f/V.r + I13522 A.f/END.r
  • linear pSB1T3 V.f/V.r + I13522 A.f/END.r

Successful assemblies should give 'green' colonies since I13522 constituatively expresses GFP under a tetR promoter.

Result (11/06/2011):

  • linear pSB1C3 only: ~ 400 colonies, ~ 50 % red (RFP) from contaminating circular parent vector.
  • linear pSB1C3 + I13522: ~ 400+ colonies, ~ 50 % red (RFP), ~ 10 % green (GFP), ~ 40 % uncoloured
  • Tet plates (pSB1T3 reactions) - lawn of bacteria .. bad antibiotic ?

Conclusions:

  • Appears that the assembly reaction worked for pSB1C3 + I13522, although at low efficiency.
  • Need to reduce amount of circular parent vector by using less template in PCR reaction of DpnI treating linear vector PCR products.
  • There are many resistant colonies that don't appear to be producing GFP (ie likely to contain no I13522 part) - possible that the linear vector is able to recicularize in the reaction through production of weakly annealing sticky ends ?
  • Should do a I13522 A.f/END.r only control, in case GFP colonies represent part template vector contamination.


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