Team:Brown-Stanford/Lab/Notebook/Week4

July 5, 2011

PowerCell

• Discovered significant (24bp) hetero-dimer between genomic DNA-targeting primer sections in all anabaena primers. The annealing temperature of the heterodimer is between 53 and 59 degrees celcius, making hybridization of primer to genomic DNA rather than other primers very difficult. We think that the E-X biobrick prefix and RBS overlap overhangs may further impede genomic hybridization, making a lack of high-affinity competitor hybridizations very important. Reexamining primers.
• Identified problem: The segment of primer g001 intended to target 400bp upstream of psaC in Anabaena instead forms the reverse complement of the section of primers g002, g003 and g004 targeting 20bp upstream of psaC. This explains the heterodimer, and the complete lack of genomic amplification. This has been designed, and will be reordered.
• Nutrient Dependency Assay experiment: repeat with sugar concentrations at those reported by Ducat at SB5--best possible was from E. coli engineered to express cscB and treated with 100mM NaCl. after 160 hours, 6mM sucrose was observed. If we repeat the experiment with this best-case, E. coli may be observed.
• Perhaps sugar gradient?
• Inoculated liquid culture of same E. coli as last time; pSB1A10-J133452
• Microscopy of first NDA experiment--no cells visible in BG11+glucose+fructose
• PCR: Nostoc primer sets (fwd g0005 with rev g0006-g0008)
• To vary annealing temperatures: used gradient PCR between 50-58C (see entry in PCR notebook)
• PCR: Primers g0015-g0017 have arrived; used g0016 and g0017 to amplify the GFP mut3b from 2011 Registry (BBa_I719015- GFP mut3b generator with T7 promoter)
• Rehydrated I719015 from Registry Plate 1/well 15P; used 0.5ul for 4x reactions
• Used four-temp gradient PCR (57.5, 55, 53.2, 51.7 C)
• Made calcium chloride competent cells (see other lab notebook)

FRETSensor

• Made 1 M stock of HEPES (LM20110705 A1)
• Made 1 M stock of MOPS (LM20110705 A2)
• Made 250 mL TfBI (LM20110705 A3)
• Made 100 mL TfBII (LM20110705 A4)
• incubated more top10

July 6, 2011

PowerCell

• ran gel of GFPmut3B amplification. No amplification. See PCR notebook.
• TOP10 competent cells transformed with GFPmut3B. Unsuccessful, although the same cells successfully transformed other plasmids.
• PCR run on Nostoc vegetative promoter with annealing gradient of 40-50?C. See PCR notebook, troubleshooting below.
• Set up and commenced nutrient dependence assay 2 with varying amounts of glucose+fructose and E. coli. See experimental design folder.
• Troubleshooting the PCR
• Possible stages of error:
• Primer interactions
• Faulty master mix
• incorrect annealing temperature
• incorrect elongation temperature
• drastically lower annealing temp to 40C
• primers: mismatched lengths is sub-optimal, excessively long flanking portion
• run blank reaction (fwd and rev primers, H2O) to examine primer interactions
• run control PCR (previously verified primers + known template) to prove reagents work, user protocol is correct
• PCR additives? http://bitesizebio.com/articles/pcr-problems-try-an-additive/
• Reorganized lab space in 378, were assigned bench space to contain mess

REGOBricks

• grew up e. coli for plasmid prep
• ran tap water through cementation column (24 hours)

FRETSensor

• Made 5mL competent cells
• Transformed with D4, [http://partsregistry.org/wiki/index.php/Part:BBa_I719015 GFPmut3b] and pbu11

July 7, 2011

PowerCell

• Ran gel of temperature graded medium RBS nostoc vegetative promoter, saw no positive control, saw amplification of primers. Faint bar on one nostoc lane.
• Perform gel extraction on the amplified band in Nostoc + mid RBS (at 50C)
• check on NDA
• Elhai will resend transformation plasmids! Possibly one with a methyltransferase!
• took micrographs
• ran positive controls pcr (see pcr notebook)

FRETSensor

• Plates with D4 and pbu11 grown (~30 colonies)
• Plate with GFPmut3b did not grow, check plasmid...

REGOBricks

• Designed primers for cloning out urease cassette from pBU11
• Began column cementation - copy down mass, volume, pore volume (approx.), etc.
  • approx 250 ml of cells (sample taken----needs to be ODed) cycled through at 11ml/hour
• Cells allowed to flow through for 16 hours after cells initially met with the column (not just sitting in the tubing)

• • • Samples OD600 with cuvette at 1.542
    • Urease activity: (measured with 5 mL cells in 30 mL of 1.5 M urea, using conductivity meter in rm 242 for 10 minutes):

cells have a urease activity of 0.40 mM urea hydrolyzed per minute, specific urease activity (divide urease activity by OD)- 0.259 mM urea hydrolyzed/min/OD

• • (Whiffen has: on the order of 10mM urea hydrolyzed/min?)

• Received new sample of S. pasteurii from Terran from SDSMT, colonies looked like what we had on the plate on black friday, we think that the colonies were actually S. pasteurii

• • Ran plasmid prep and RNA digest on HB101 cells containing pBU11, unfortunately misplaced undigested plasmids. Ran gel electrophoresis on the digested samples.

• Wells

1. Testtube 1: plasmid prepped using Norman’s digest
2. Testtube 2: prepped using Norman’s digest
3. 1kb DNA ladder
4. Testtube 3: prepped using miniprep kit
5. Testtube 4: prepped using miniprep kit
6. 1kb DNA ladder

July 8, 2011

PowerCell

• PCR of gel-extracted nostoc vegetative promoter
• Gel of positive control-o-rama
• Continued cryostocks
• took lots of micrographs
• Gel of nostoc gel extraction
• lab meeting presentation!

REGOBricks

• Inoculated 2x 10 mL and 1x 5mL LB/amp liquid cultures of HB101 for
• plasmid prep
• urease activity

• Inoculated 1x 75 ml liquid culture Bang liquid culture of S. pasteurii for
• Have 2x LB + amp E. coli plates
• Measured specific urease activity of actively growing S. past
  • OD:
  • Urease by Cond:

A*t 10:20 (after 16 hours of bacterial pump), calcium chloride was introduced into the setup and column, at same rate (11ml/hour)

• Restriction enzyme digest protocol:
  • 1 ul 10x restriction buffer (in box)
  • 6.5 ul H2O
  • 2 ul DNA
  • 0.5 ul enzyme
  • incubate at 37 C waterbath for _____ minutes.

• Tubes 2 + 4 were digested for 90 minutes
• Tubes 1+3 were digested for 60 minutes

• Plasmid Extraction #2. 104 volts. 40 minutes.

1. ladder
2. Norman’s control - no digest (
3. Norman’s digest (#1) -concentration: 825.8 ng/ul
4. Norman’s digest (#2) -concentration: 1.6 ng/ul
5. Mini prep control
6. Mini prep digest (#3) -concentration: 8.8 ng/ul
7. Mini prep digest (#4) -concentration: 1.6 ng/ul
8. Ladder

• Nanodrop
  • Protocols:
    • blank with 1 uL of media (EB for miniprep, TE for norman)

July 9, 2011

REGOBricks

• Took Biosensor's top10 transformants from incubator

• Cementation column trials:
  • Spun down cells (OD 1.872)
  • Resuspended in fresh urea broth and 1:1 M CaCl2
  • Added to 5 and 25 g samples of lunar regolith simulant JSC-1

• Dishes

FRETSensor

• Plated out GFPmut3b