Team:KULeuven/2 September 2009

From 2009.igem.org

Project progress

Progress of parts

[edit] Blue Light Receptor

  1. FACS measurements:
    • The cultures from shift 2 had similar results as shift one. However, the GFP signal measured from the LigA construct was stronger. Thus, cells definitely need to grow before undergoing irradiation.
  2. The dilutions and the shift one cultures put overnight did not show any significant results.
  3. BBa_J23101 was miniprepped and nanodropped
Part concentration (ng/μl) 260/280 λ
BBa_J23101 (A) 238,9 1,95
BBa_J23101(B) 248,8 1,91
  1. After nanodropping, BBa_J23101 was restricted with SpeI and PstI, while BBa_E0240 was cut with PstI and XbaI.
  2. A gel electrophoresis and extraction was performed. This showed an unexpected 800 bp signal at the lanes with BBa_J23101. This is in fact due to the vector in which this part is ligated. BBa_J61002 contains a reporter gene, RFP. Thus, in combination with the promotor, a construct is formed that can be used to measure activity of this promotor. Since the promotor is flanked by standard assembly restriction sites we can replace the current promotor (BBa_J23101) by any other promotor, for instance BBa_K238013.
  3. DB3.1, BBa_J23101 and LigA were ented in liquid culture and grown overnight to make glycerol stocks and competent cells

[edit] Vanillin Production

  • The four grown colonies are put on liquid culture
  • New (again...) restriction digest on sam5, sam8, fcs and ech, genes were miniprepped on Tuesday.
    • Overnight at 16°C
  • The plates with sam5, sam8, ech and fcs were re-ented. From sam5 and fcs four colonies were picked and plated.
  • The PCR from yesterday did not deliver any results, probably because of an error when preparing the PCR mix. A new PCR was prepared and started today.

[edit] Vanillin Receptor

  • there was made a controllgel of virA for our basicproduct, purification and nanodrop
  • the two ligations X+K were checked and electroporated
  • X was digested again and ligated in K
  • There were fluid cultures made for ligation of Y+K
  • A new PCR was done for rpoA mutagenesis : elongationstep was now taken 4 minutes instead of 1 minute

[edit] Key/Lock/Anti-Key