Team:Brown-Stanford/Lab/Notebook/Week13

September 5, 2011

PowerCell

• Very small speckley colonies on the preliminary balloon revival plates. Don’t really look like cyanos, but maybe?
• Microscope these!
• Transformed Ana-GFP ligation
• made 2 plates, 300ul cells to each
• Liquid cultured CHK1.1,3 CHK2.1-6, GPFN

September 6, 2011

PowerCell

• miniprep of liq cultures from 9/5
• ordered sequencing of minipreps (18 total rxns)
• Lots of Ana-GFP transformants! About 20 on each plate! Yayyyyy!
• inoculated 3 liquid cultures from each (10 mL cultures, in 15 mL falcon tubes), w/ LB+chlor
• labeled x.y.z, where x=attempt, y=plate, z=colony
• Make BG11 + 5%LB + 1% agar
• Microscope balloon test revivals
• if good, prep balloon flight cyanos

REGOBrick

• Gel extractions: Approx 50 μL of 17.4 ng/μL = approx 850 ng product
• Made mini bricks
• made quick brick
• Immobilized S. pasteurii on nitrocellulose for flight:
  • Ground control:
    • 2x left open to the air
    • 2x contained in whirl-pak bags
  • Flight (inside):
    • 2x left open
    • 2x in whirl-pak
  • Flight (outside):
    • 2x left open
    • 2x in whirl-pak
• Made urease plates to test urease activity afterwards, have bang plates on which to grow retrieved specimens.

September 7, 2011

PowerCell

• miniprep AnaGFP biobrick candidates
• 1.1.1 133ng/μl
• 1.1.2 101 ng/μl
• 1.1.3 89 ng/μl
• 1.2.1 113 ng/μl
• 1.2.2 98.5 ng/μl
• 1.2.3 121 ng/μl
• sent in for sequencing
• check sequence data from 9/6
• GFPN good!
• all of the cscB candidates are a bust
• most ended up being the lux brick
• figure out what to do about cscB
• PCR cleanup on the digest
• because it had both taq PCR and digest crap
• digest of Ana biobrick as an alternative source of pSB1C3
• also ordered plain pSB1C3 from the registry
• used AHK5 because AHK4 was empty
• inoculate new cell culture of our primary Ana biobrick source (AHK4) for cryostocking

REGOBrick

• Plan for the next couple of weeks in order to brick and submit our urease part:

1. Restriction (edited Knight lab protocol)
   1. 5 μL NEB2 buffer
   2. 25 μL DNA (17 ng/μL)
   3. .5 μL of 100x BSA
   4. 1 μL of enzyme 1
   5. 1 μL enzyme 2 - NOTE for both of the restriction enzymes, you need to be careful about pipetting; the enzymes are stored in glycerol which can stick to the outsides of the pipets. So when pipetting, only touch the tip to the surface of the water.
   6. 17.5 μL ddH20

• Add water to .6 mL tube
• Add restriction buffer (vortex beforehand to ensure mixing)
• Add BSA (vortex)
• Add DNA (vortex)
• Add enzymes
• Place in thermal cycler
  • 4-6 hours incubation @ 37 deg
  • Go directely to cleanup with Wizard SV PCR and Gel cleanup systems

• Vector digest - Silver lab
  • 700 ng vector
  • 1 μL of 10x BSA
  • 1 μL of appropriate buffer for the selected restriction enzymes
  • .2 μL enzyme 1 (20 units/μL)
  • .2 μL enzyme 2
  • Distilled H20 to total volume
• Incubate at 37? C
• Next morning add .1 μL CIP and incubate at 37 1H (?)
• Go to cleanup?

• Ligation
  • x μL vector (20ng)
  • y μL insert (1:1 insert to vector ratio or 3:1 insert to vector ratio? if 1:1, 103.38 ng of insert will be needed. if 3:1, then approx 310 ng will be needed.)
  • 1 μL of quick ligase

• Add appropriate amount of water
• Add ligation buffer (vortex before pipetting)
• Add insert
• Add vector
• Add 1 μL ligase - vortex ligase and watch for the glycerol sticking
• Incubate for 5 min on benchtop
• Place on ice until transformation
• generally 1 μL of ligation mix is sufficient
  • Optional: heat inactivate at 65? for 20 in and then go straight to transformation... no need for 20 min

• Transformation with k12 cells

September 8, 2011

PowerCell

• balloon launch!

REGOBrick

• Filming with the BBC crew

September 9, 2011

PowerCell

• cryostock Ana biobrick (from colony AHK4)
• miniprep more Ana biobrick -> 185 ng/μl

REGOBrick

• 50 μL PCR to obtain more urease cassette (successful protocol)
• We will be getting pSCB1C3 + J04450 in the mail from iGEMHQ
• Waiting on sequence data to determine if there are any restriction cut sites in our operon. If so, we need to restrict at NOT those sites or do site directed mutagenesis.