IGEM:MIT/2007/Notebook/2007-7-16

F2620 Expression Protocol

1. Create Liquid Cultures in LB Media
2. Dilute Back 1:500 in LB Media
3. Grow up for an hour in warm room roller
4. Split culture in two
5. Add AHL to one culture to a final concentration of 1E-6M
6. Grow up for 4 hours in warm room roller
7. Spin Down, Decant, and Resuspend in PBS
8. Measure absorbance at 600nm and red fluorescence level

See characterization data for BBa_F2620.

Today's Agenda

• Redo Overgrown Liquid Cultures
• Send for Sequencing
• Cut CPX and Backbone for ligation
• Start designing high copy backbone
• PCR off relevant portion of Mer operon from pto40 - Done
• Organize fridge - Done
• Design and order EC_20 Insert
• Email Isadora + Tom Knight for Meeting Times--Toan--Done
• Anneal FHUA Insert oligos - Done
• Understand Meinke Inducible System

Status of Expected Parts

1. IDT Primers + Inserts - Arriving Today--ARRIVED
2. CPX - At FedEx Facility in Boston
3. FhuA - ARRIVED

Sequencing of LC#1&2 from 3A

• Miniprepped LC's of those colonies that grew only on Kan plates and not on Kan/Amp plates
• Sent in 4 samples, one forward and one reverse for each LC
• used 4µl of DNA since each was approximately 30ng/µl
• Order Number 8046

Dilution of Ordered DNA

• Made 100uM stocks of all IDT primers and single-stranded inserts
• Diluted 100uM stocks of primers/inserts into working stocks of 10uM concentration

Annealing Complementary Primer Inserts

• 10 uL of 10 uM of oligo
• 2 oligos - total volume 20 uL
• Mix with 80 uL water
• PCR Thermocycler Temperatures
• 95 C
• Ramp down
• 0.1 C/sec

Phosphorylation of Primer Inserts

• 20 ul Annealed Oligo
• 4 ul T4 DNA Ligase Buffer
• 2 ul PNK (Kinase)
• 14 H20
• 37° - 1 hr, 65° - 20 mins

Digestion of FhuA backbones

• used PCR Purification kit to purify backbone

Nanodrop of FhuA inserts/backbone

• PICCS5 - 10ng/ul
• PHIE6 - 5ng/ul
• Insert- 650 ng/ul - instead just use rule of thumb .5 ul/20ng backbone

Ligation of FhuA inserts/backbone

• Mix 1: PHIE6
  • .5 ul Ligase
  • 2 ul Ligation Buffer
  • 1.5 ul Insert
  • 10 ul Backbone
  • 6 ul H20
• Mix 2: PICSS5
  • .5 ul Ligase
  • 2 ul Buffer
  • 1.5 ul Insert
  • 5 ul Backbone
  • 11 ul H20
• Program 16IGEM