Team:KULeuven/15 September 2009

From 2009.igem.org

Project progress

Progress of parts

[edit] Blue Light Receptor

  1. miniprep of pBR322 (nanodrop: 24,5ng/µl)
  2. restriction digest of pBR322 with EcoRI and PstI
  3. gelelectrophoresis and extraction of pBR322 (nanodrop: 10,8ng/µl)
  4. ligation of pBR322 with ligA (blp + BBa_E0240)
  5. electroporation of cells with LigY (attempt 3)

[edit] Vanillin Production

  • Restriction SAMS1 1 X/P, BBa_J23109 S/P, EF1 and EF2 E/S, COMT4 2 and COMT1 1 E/X
    • SAMS and EF show expected results, promotor shows the same result as yesterday (1 part of 900bp and 1 of 2k bp) and COMT looks good.
  • Ligation of EF + TER, EF was not purified from gel, instead the enzymes were heat inactivated on 65C for 15 min, TER was an older vector which was purified
  • Promotor BBa_J23109 transfered from glycerol (-80) to a plate and liquid culture.
    • At this point we realised the promotor was not in a standard vector but rather in BBa_J61002 which contains an additional RFP after the Spe restriction site, thus a part of 900bp and one of 2k bp is expect when digesting with P
  • Restriction COMT2, 3 overnight.

[edit] Vanillin Receptor

  • Because virA was now properly inserted in the pucvector we could now start mutagenesis on restriction sites 187 and 720. We did this with PCR. We used a protocol for site-directed-mutagenesis with the matching primers. PCR was done overnight
  • new ligation for virB was done

[edit] Key/Lock/Anti-Key