Team:KULeuven/3 September 2009

From 2009.igem.org

Contents

Project progress

Progress of parts

[edit] Blue Light Receptor

  1. A restriction digest on BBa_J23101 with EcoRI and SpeI was performed. This will cut the promotor out of its vector (BBa_J61002) which has a RFP reporter gene. We will put BBa_K238013 (cut with EcoRI and SpeI) in this vector. This way we will get a construct where RFP will be produced dependant on our blue light promotor. We can then compare this with the intensity of RFP produced by BBa_J23101.
  2. Glycerol stock was made from LigA and BBa_J23101. They are stored in the -80°C in S&P F8 67-68 and 69-70.
  3. Ecoli strain DB3.1 cells were made competent for electroporesis.
  4. Gel electorphoresis and extraction on BBa_J23101

[edit] Vanillin Production

NOT PCR

  • Electroporation of SAMS+TER ligation (from Tuesday ligation... the one that looked bad....)
  • Miniprepped the 4 colonies from SAMS
part concentration 260/280 260/230
SAMS 1 35,4 1,97 1,18
SAMS 2 49,3 2,00 0,97
SAMS 3 62,0 1,97 2,65
SAMS 4 78,3 1,85 1,43
Terminator 181,4
  • Restriction digest of SAMS
  • Gel electrophoresis of restriction from ech, fcs, sam5 and sam8
    • Result looks great (woohoo!)
    • Cut and purified from gel
gene concentration 260/280 260/230
sam5 8,2 1,62 0,02
sam8 1,4 3,04 0,01
ech 10,1 1,50 0,05
fcs 5,2 1,61 0,03
  • Used ech and fcs to ligate using 5 μl from ech and 23,5 μl from fcs. There was not enough sam8 for ligation. Redo restriction at some point in the future...
  • 4 colonies from sam8 and ech were picked and plated

PCR

  • The PCR from yesterday went very well, we had a huge amount of amplified biobrick DNA of the different parts. Agarose gel electrophoresis was used to test if the different pieces of DNA created by PCR had the correct length.
gene concentration 260/280 260/230
sam5 361,6 1,91 2,13
sam8 323,8 1,92 2,06
ech 236,5 1,91 2,24
fcs 321,4 1,90 2,22
  • Amplified DNA was purified and cut with different restriction enzymes. After restriction, DNA was purified again before ligation.
gene concentration 260/280 260/230
sam5 21,8 1,65 1,33
sam8 17,1 1,56 1,16
ech 13,8 1,78 1,46
fcs 20,9 1,70 0,20
  • After the restriction, sam5, sam8 and terminator were ligated in a three-way ligation. The same was done for ech, fcs and terminator. We also started a ligation of just sam5 and sam8 and ech and fcs.

[edit] Vanillin Receptor

  • The Y+K ligation was miniprepped and nanodropped to make later on a stock on glycerol
  • We decided to use for virA a puc19-vector in stead of TOPO, puc was digested with Xba1 and Ecor1 and virA was digested with EcoR1 and Spe1. This was done overnight.
  • electroporation + plating was done for X+K

[edit] Key/Lock/Anti-Key