Team:Cambridge/Notebook/Week4

Monday

• Wet work
  • Transformed competent cells using plasmid phK724 from Jim Slock (thanks!)
  • Grew up E. coli containing phK555 in 5ml LB broth in rotating 37°C incubator overnight
• Dry work
  • Finalising sequencing requirements
    • Dismissed PPK
    • Decided to include Luciola cruciata
  • Further Wiki work

Tuesday

• Wet Work
  • Made competent cells of 4 different hns mutants: W3110 hns93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10, GM230 hns-205::Tn10
  • Now stored in the -80°C freezer for later use

• Dry Work
  • Organising sequences for synthesis

Wednesday

• Hannah and Theo tested previous day's preparations for competence
• Peter and Paul transformed phK555 with phK724, placed cells in 30C incubator (above rotating incubator)
• Bill, Emily, Anja performed miniprep and restriction digest then ligation.
  • Decided that we may have used wrong technique, but DNA samples in fridge

Thursday

Results

GM230 hns-205 ::Tn10 TetR (black) - somewhat competent W3110 hns 93-1 (blue) BW25113 Δ hns ::kan (red) W3119 hns-205::Tn10 Tet^R

Theo's plan

1. Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol
2. Digest luciferase Biobrick with: XbaI, PstI to produce two fragments:
   1. P end---old backbone---E--Xend
   2. X_end--luciferase---S--P_end (desired)
3. Digest backbone Biobrick with SpeI, PstI to produce:
   1. S_end--short fragment--P_end
   2. P_end---backbone--E--X--promoter--RBS--S_end (desired)
4. Run both digest products on a gel
5. Extract suitable lengthed fragments from gel:
   1. Promoter + backbone should be 2153 bp
   2. Luciferase should be 1653 bp (its 3426 bp backbone should be discarded)
6. Perform ligation
   1. S and X are compatible so we get
      P_end---backbone--E--X--promoter--RBS--SX_scar--luciferase---S--P_end
   2. The two P_ends will also ligate, producing a circular desired plasmid
7. Transform competent TOP10 cells with ligation product

Friday

Saturday

Sunday