Team:Brown-Stanford/Lab/Notebook/Week0

• REGObricks
  Intro --------------------- ISRU --------------------- Biocement --------------------- S. pasteurii --------------------- Balloon
  Flights --------------------- Transforming --------------------- Biobrick²
• PowerCell
  Intro --------------------- Cyanobacteria --------------------- Mars --------------------- Nutrients
• FRETcetera
  Intro --------------------- FRET etc. --------------------- Construct
• Lab
  Team --------------------- Parts --------------------- Notebook --------------------- Protocols --------------------- Safety
• Partners
  Collab --------------------- Sponsors
• Outreach
  SB5.0 --------------------- NASA --------------------- Lunar Science --------------------- Maker Faire --------------------- BBC --------------------- CBricks --------------------- Ethics --------------------- Regional
  Jamboree

June 6th, 2011

FRETSensor

We spent the morning listening to presentations from researchers in Building 239, Lynn’s colleagues. The following is a summary of their research and expertise, which may be useful to consult throughout the summer:

• Chad Paavola: cellulosome complex, cellular biosensors
• John Cumbers: chrococcidiopsis, UV radiation resistance
• Matt (from Marshall Space Center): CO2 reduction systems; phys/chem. perspective
• Darya Bubman: used to be a molecular viologist, working in synth bio group
• Sigrid Reinsch: (worked in Shank’s lab!) OMEGA offshore bioreactors, freshwater algae in wastewater
• Rocco Mancinelli: bioreactors, nitrogen cycling, designing bioreactors that can convert purposes
• Lynn Rothschild: overview of lab researchers:

       	**Kosuke: RNA expert, pro mol. Biologist
       	**Ivan: DNA dmg in Mn complexes
       	**Haley: biosensors
       	**Diana: air capture devices
       	**John Cumbers, Norman Wang (from Hawaii 08 team): chrococidiopsis, evo, syn bio
       	*Thomas Beer, Lynn: cyanidium, snow algae, UV resistance

• • David Loftus: MD PhD; effect of radiation (UV, solar wind, GCR, SPE protons) on regolith, effect of regolith on human physio;
  • John Hogan: biological life support systems; ecosystems, closed; regenerating life support systems in space

June 7th 2011

FRETSensor

We presented an overview of iGEM and our projects to NASA researchers: Chad, John, Sigrid, Matt, Darya, Lynn, David Loftus

In the afternoon, we presented to Pete Worden (ARC director), Gary Martin (Director of Strategic Management and Advanced Planning). They have offered to assist us in any reasonable capacity

Team pictures with Director Worden, all present! (except Eli :( )

June 8th 2011

Minor organizational emails and administration in the office, spent the entire afternoon attending lab safety bootcamp. All members are now educated on prudent laboratory practices and can safely handle research in the lab!

June 9th 2011

• Eli completes his EAP introduction class/forms

PowerCell

• News of many strains of our cyano strains will be sent/are in transit
  • Nostoc punctiforme
  • Cystis 6803
  • Synechococcus elongatus PCC7942
  • Anabaena PCC7120
• Requisite tools and instruments were placed in acid bath overnight in order for media production on friday.

FRETSensor

• Met with Dr. Chad Paavola to discuss our plans for the DNA damage sensor. Our original idea would have relied on fluorescent protein reporters, a system whose function would have been too slow to be useful. Dr. Paavola instead suggested a cohesin-dockerin system in which fluorescent proteins, linked with dockerin domains, are constitutively expressed. Upon UV radiation and LexA/SOS pathway activation, linked cohesin domains are expressed. Once present, these cohesin domains bind to the dockerin subunits of the fluorescent module, causing the fluorescent subunits to become excited and generating a visible signal.
  • This system could react much faster to LexA/SOS pathway activation, as the flourescent proteins are constantly expressed and available, and only need to be activated by the cohesin domains.

June 10th 2011

• Evan arrives!
• Discuss directions for DNA damage sensor project:
  • It seems as if the first objective is to troubleshoot Ryan and Evan’s constructs from BioE44; this is the “sensor” portion of the experiment, and would demonstrate the viability of the SOS pathway as a sensor
  • FRET and cohesin/dockerin complex has now been split off into a separate sub-project to make the actuator half of the sensor
• Learned how to make LB, BG-11, and S. Pasteurii culturing media in the lab

June 11 2011

• Picked up “DNA Damage sensor with no LexA Generator” that Evan and Ryan built in Stanford BioE 44 from Drew Endy’s lab as well as preliminary experiments run on that construct.
• Developed a preliminary timeline for our wastewater screen for urease activity and potential urease genes.

June 13 2011

• Intern/student orientation and reception
• DNA damage team researched fast fluorescent reporters, made timeline for project
• Media made for S. pasteurii with uv hood and filter sterilization. Did not autoclave each ingredient separately.
• Solidified relationship with the Bang lab, who will be sending us S. pasteurii, PBU11 e coli, and the urease-coding PBU11 plasmid. Good stuff.

June 14 2011

• Cementation: made tentative schedule for chemostat, brick-making constructions.

June 15 2011

• First day of SB5.0 at Stanford. Goes through 17th.
• First parts submitted to registry: cohesin-gly-gly-ser-gly-cohesin, dockerin-CFP, and dockerin-YFP

June 16 2011

• Received package from the Bang lab, materials from Sigma have arrived! Materials were put in 4.5 degree refrigerator, will make media/plate them tomorrow.

June 17 2011

• Opened package from the Bang lab, contained 2x agar stabs of B. subtilis, 1 plate of S. pasteurii, 1 plate of E. coli with pBU11 plasmid (S.p. urease gene), and 1 tube of pBU11 plasmid (amp resistance); agar stabs of pURE91 (B. subtilis urease gene) must grow up overnight liquid cultures before making glycerol stocks:
  • LB amp+ for E. coli with pBU11, S. pasteurii media @ pH9, and B subtilis???
• Four strains of cyanos from the Golden lab (duplicate tubes of each) have arrived in liquid culture: add 1ml of culture with 3ml of BG-11 in a 5ml culture tube, place in 30C incubator with tissue covering light
• For Norman: there are six tubes of LB in agar stabs to streak out; some have Kan resistance, others have Amp (KM20, RP1 = kanR; pUC18, pSB1A3 = AmpR); don’t touch the one labelled pRL1383a
  • to streak, make Kan+ (20 ug/ml) and Amp+ (100ug/ml) LB plates; use sterile toothpick to place drop on each plate, then close bacteria and return to fridge; use a second toothpick to streak each plate
• Due to lack of autoclave, NOTHING ACTUALLY HAPPENED