Team:Brown-Stanford/Lab/Notebook/Week3

June 27, 2011

PowerCell

• Finished and ordered Gibson primers
• took out native RBSes from 400bp upstream promoter regions by chopping last 20 bp
• fixed 20 bp overlap problem
• had only 20 bp overlap, needed 40
• Only amplifying to the first stop codon of the cscB gene - this is what the Varela paper uses for their sequence
• used norman’s improved prefix and suffix
• Some of them form hair pins, and are way long. We’re worried especially about the 179 bp cscB-terminator-SP one. Best to just test and see says norman.
• Also order restriction enzymes, verification primers
• Requested cscB strain or plasmid from Silver, Varela
• Silver says no, its unpublished work
• waiting to hear back from varela
• Created BG110 (N-) and BG110 + 200μM Glucose for co-culture test
• Refined co-culture experimental design
• Attempted cell counting with Anabaena with hemacytometer
• cells are filamentous, perhaps disrupting random distribution across array
• cells are tiny
• Experimented with reading OD with spectrophotometer and calculating cell density in preparation for innoculation cell count in co-culture test

June 28, 2011

PowerCell

• Discarded first attempt to revive Silver’s S. elongatus
• Attempted three more liquid cultures of Silver’s S. elongatus
• Received word from Varela that he will send cscB E. coli
• Prepared co-culture media, revised experiment design
• Innoculated 3ml of LB amp with E. coli in preparation for sugar/nitrate culturing tests
• E. coli strain: Norman’s pSB1A10 w/ J133452 (contains RFP reporter)
• placed in 37C at 4:10pm
• Innoculated 100 mL BG11 liquid stocks of Golden Strains
• placed in 37C around 4pm
• made fructose and sucrose for NDA (nutrient dependency assays)

FRETSensor

• Started growing cells for Julius’s fast competence/transformation protocol

June 29, 2011

PowerCell

• All the primers from Elim have arrived!
• Commenced NDA experiment
• Measured results of plasmid prep with nanodrop (see results in Plate/Sample reference book in Notebook folder)
• Prepared 100ml cyano stocks for cryopreservation

FRETSensor

• Completed most of the protocol until we forgot LB media for the cells and they died screaming in the incubator in only CaCl2

REGOBricks

• Established contact with Eduardo Nicolau-Lopez
• nicolaulopez@gmail.com
• He is attempting to build a bioreactor pathway to hydrolyze urea purified from urine, and then use the ammonium to power an alkaline bioreactor

• Max:
  • Microscope slide experiment
    • 10 uL of cells (OD600 of 0.396 nm)
  • Made 1L of Bang media aliquots of
    • 2x 15 mL for cuvette sampling
    • 8x 50 mL in falcon tubes
    • remaining in giant filtered tube
• Ryan:
  • Grew 6 liquid cultures from liquid stock (100 mL)
  • Grew 6 liquid cultures from glycerol stock
  • Plated liquid culture on Bang plate
  • Plated glycerol stock on Bang plate
• Andre:
  • Bought new tubing for bioreactor
  • bioreactor design: peristaltic pump pulls the tubing if too large (3/8 inch), works much better with 1/4 inch. Also, flow rate has been established.

June 30, 2011

PowerCell

• Inoculated liquid culture of Sherman’s Cyanothece 51142
• Identified sucrose-utilizing E. coli strain W, ordered from ATCC as demonstration of sucrose uptake from cyanobacteria.
• Decided to make additional fluorescent reporter/cscB construct to test vegetative promoters
• designed the 3 new primers for gibson assembly of reporting construct
• re-checked and ordered reporter primers
• Amplified Nostoc and Anabaena Promoter sequences out of organismal genomic DNA with E-X prefix and RBS-overlap
• PCR: used 53C annealing temp, XXX seconds on elongation step, XX cycles
• Ran product of PCR out on 1.2% agarose gel, 35min @ 96V;
  • Lane 1 (from top): Ana + weak RBS (10ul)
  • Lane 2: Ana + mid RBS (10ul)
  • Lane 3: Ana + str RBS (10ul)
  • Lane 4: 100bp ProMega ladder (10ul) - too much?
  • Lane 5: Nos + weak RBS (10ul)
  • Lane 6: Nos + mid RBS (10ul)
  • Lane 7: Nos + str RBS (10ul)

• Gel imaged on UV Transluminator, and Typhoon in Lab 335 (using fluorescence, blue excitation)
• Ladder showed strong smearing. Only bands in lanes 5-6 (suspected above the 500bp ladder)
• Next time: use less DNA ladder (, 15ul of PCR product, 2% agarose gel, green excitation
• NDA experiment: Nothing seemed to grow. Also, 3 of the culture tube caps fell off during shaking incubation, so those are possibly contaminated. Noted which ones these were on the tubes

REGOBricks

• Ryan:
  • All 6 10mL cultures from liquid stock are turgid
  • One of the 6 10mL cultures grown from the -80C glycerol stock grew
  • Plate grown from liquid culture grew
  • Plate from gylcerol stock did NOT grow

• Plated S. Pasteuri at dilutions of 1x, 10x, 100x, 1000x, and 10000x @ 11:15. Done in conjunction with OD reading to find preferred OD for colony growth.

• Liquid cultures from Glycerol stock were turgid in the afternoon

• andre:
  • bioreactor flaws:
    • air bubbles
    • water/nutrients need to percolate up in order to more easily get rid of air in the column
    • conducted test with very fine sand, and a potential issue came up. Once water has saturated fine powder like lunar dust, it is very difficult to push an air bubble through it again. Therefore, once the dust is immersed, no bubbles can be introduced into the system. Need valves.

• Max:
  • made ghetto-bricks
    • inserted fine sand into 4the small tubes, added concentrated cells and cementation fluid (.75ml each)
    • left overnight (room temp), then thrown into 30 deg incubator.

• Check on microscope slides

July 1, 2011

• Came in the morning, 4C fridge in lynn’s lab was sounding alarm. Temperature: -13.5C. Everything inside is frozen.
• Discarded all plates and rethawed media. Nothing broke.
• Made new Amp plates

PowerCell

• aliquoted gel into two 100ml batches, increase agarose concentration in one to 2% from 1.2% by adding 0.8g agarose.
• Attempt 2 of the vegetative promoter PCR and gel from yesterday, with some modifications. See PCR notebook for details. Raised extension temperature to that dictated by Promega for the polymerase (72?C from 68?C) and raised the annealing temperature to 55?C. Saw very faint bands at the desired size (~400bp) for two of the nostoc products, otherwise only bands at the 100-200bp range for all products. These are the same bands that were visAnnealing temperature back to what it was for the next run?
• Still no visible growth in the NDA experiments.
• Designed primers to add reporter to cscb vegetative promoter construct; Reporter is a GFP mut3b found in the registry (optimal for detectable fluorescence despite cyano pigments)

REGOBricks

• Black friday: came into lab to check on new cultures and dilutions; a lot of the colonies looked odd, and we feared contamination
  • made all new plates (bang rich media)
  • researched pbu11 urease genes
  • counted and took pictures of e coli on slides (see ryans email)

July 2, 2011

REGOBricks

• microscope slide pics of coverslip experiment
• grew up urea plates
• Constructed boxes and column for actual bricks
  • Column: 50 mL falcon tube
    • ? inch flexible plastic tubing
    • Peristaltic pump
    • Scotch brite
  • 50mL falcon tube bottom was cut off and attached to tubing with plastic polymer glue. The top of the tube was left open, and a plastic stopper was fitted into the top. Another tube of the same size was fitted through the stopper.
  • Liquids (water first, then cells, then cementation fluid) will be pulled trough the bottom of the column and out the top through the negative pressure supplied by the perstalitc pump, which works on the outlet tube coming from the top of the column.
  • 51.1625 g of JSC-1 lunar simulant was loaded into the column and packed to approx 35 mL. Look at the JSC-1- paper again, but the porosity of such a sample is approx 9-10 ml.
  • Worked on peristaltic pump, found a setting that allows for 11 mL/hour movement.