Team:Penn/Notebook

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Penn 2012 iGEM Wiki

June 2012 Notebook

Week 1

June 6th

  • Set up some lab equipment
  • Autoclaved for a while
  • Organized biobrick stuff
  • Called Vinoo about DNA planning

June 7th

  • Transformed Cph8, pLsr, and LuxS
  • Placed order with Vinoo
  • Developed idea using PGY/PCN system to activate a gene

Week 2

June 11th

  

Wet Lab

  • PCR'd mCherry from NAS157
  • Ran 1% Gel and purified product
  

Dry Lab

  • Designed primers for LsR promoter
  • Meeting with Dr. Sarkar

June 12th

  

Wet Lab

  • Digested mCherry PCR product with BamHI and NotI
  • Column purified mCherry and ligated into NAS152 backbone
  • Transformed NAS152-mCherry into DH5alpha
  • Poured 25 LB-Kan plates
  

Dry Lab

  • Research more information about bacterial drug delivery system
  • More research into biofilm project

June 14th

  

Wet Lab

  • Fill in later....
  

Dry Lab

  • Met with Dr. Goulian, obtained pDawn and pDusk
  • Identified inaK as a surface display gene we can use

Week 3

June 18th

  

Wet Lab

  • Miniprep pDawn and pDusk
  • Test cut pDawn and pDusk with XmaI, analytical gel was correct
  • Prep cut pDawn and pDusk with BamHI and NotI, gel purified
  

Dry Lab

  • Ordered and picked up PCR purification kit from cell center
  • Additional orders through cell center
  • Designed primers for one of Peter's components (forgot which)

June 20

  

Wet Lab

  • Picked 2 colonies of pDawn-mCherry, innoculated in 5 mL of LB and 50 ug/mL of Kan
  • PCR purified fragments (Peter), then ran gel?
  

Dry Lab

  • Researched DARPin binding domains and linkers
  • Finalized some biobrick orders
  • Finalized synthesis order (minus linker)

Week 4

June 22

  

Wet Lab

  • Ashwin - Repeated miniprep on pDawn and did test cut
  • Peter - Miniprepped pet26b and digested with BglII and EcorRI
  • Avin - Miniprepped pet26b, made glycerol stock and digested with BamH1 and Not1
  

Dry Lab

  • Avin - Finalized and sent in synthesis order (still awaiting order confirmation)

June 25

  

Wet Lab

  • Ashwin/Avin - Column purification, ligation, and transformation of Pet26b-mCherry
  • Peter -run gel of eGFP/plsr ligation
  

Dry Lab

  • Avin - Sent in final gene synthesis order
  • Mike - reviewed pDawn protocol, reviewed TetR sequences
  • Peter- Order restriction enzymes from cell center

June 26

  

Wet Lab

  • Avin - Picked colonies for Pet26b-mCherry and pJT106b
  • Mike/Ashwin - Plated pJT122
  

Dry Lab

  • Everyone - Brainstormed human practices, Wiki design

June 27

  

Wet Lab:

  • Miniprepped pet26b-mCherry and pDawn-mCherry
  • Test cut pet26b-mCherry with HindIII and ClaI (picture temporarily saved in MEAM folder of pqiao account), bands looked okay, chose C2 for remainder of experiments and made a glycerol stock, transformed both pet26b-mCherry and pDawn-mCherry into BL21(DE3)- Gold (10 ul cells, ~1ng DNA) Plated 200 ul and 20 ul
  • Inoculated colonies for pJT122, pJT106b, PhyB (BBa), PIF3 (BBa), and Cph8 (BBa)
  

Dry Lab:

  • Started exploring possible wiki designs and coding

June 28

  

Wet Lab:

  • Miniprepped pJT122, pJT106b, PhyB, PIF3, Cph8, made glycerol stocks
  • Picked colonies for pET26b-mCherry and pDawn-mCherry, innoculated, grew up, then diluted Measured OD along the way
   

Dry Lab:

  • Set up light and dark incubators

June 29

  

Wet Lab:

  • Tested pDawn-mCherry and pET-mCherry, pDawn-mCherry expressed mCherry only after light induction
  • Nanodropped pJT122, pJT106b, PhyB, PIF3, Cph8
  • Performed digest of pET-26b, eGFP, and plsr
  • Spread Biobrick shipment on Amp plates (lsrR, lsrK)
  

Dry Lab:

  • Opened Bio-Rad shipments

July 2012 Notebook

Week 5

July 2

  

Wet Lab:

  • Transformed ho1 and pcyA BioBricks
  • Peter: ligations
  

Dry Lab:

  • Contacted labs for JT2 and pPL-PCB
  • Worked on Human Practices

July 3

  

Wet Lab

  • Nothing to be done for drug delivery
  

Dry Lab

  • Avin - digital schematic, helped with primers, read human practices bacterial therapy stuff
  • Mike - talked to Dan, developed cloning strategy for getting Cph8 into the pDawn backbone, started primer design

July 5

  

Wet Lab

  • Picked colonies of ho1 and pcyA
  • Inoculated pDawn-mCherry cells for timecourse
  

Dry Lab

  • worked on primer design/cloning strategy and talked to Dan
  • worked on schematic and wiki
  • worked on human practices

July 6-7

  

Wet Lab:

  • Took pDawn-mCherry time course
  • Sterilized incubator and TC hood
  • Moved lab stuff to small room
  • Redesigned primers for plsr & egfp
  

Dry Lab:

  • Contacted FDA contacts for human practices

Week 6

July 9

  

Wet Lab:

  • Inoculated 50mL cultures of pDawn-mCherry
  • Set up pH meter, picked up JT2
  • Organized lab area
  • Made bacterial streaks for biobricks
  

Dry Lab:

  • Ordered mammalian cell culture media
  • Finalized wiki template
  • Worked on cloning steps for cph8

July 10

  

Wet Lab:

  • Did time course for pDawn-mCherry, but since it was from a glycerol stock, saw no growth → will let grow overnight, then dilute in the morning and start a new time course
  • Picked colonies from transformed Biobricks and innoculated
  • Resuspended ClyA and LuxS IDT DNA and transformed into DH5alpha
  • TC/Sterile practice training
  

Dry Lab:

  • Ordered primers for ClyA-RFP, Peters primers
  • Worked on wiki
  • Emailed Tabor for pJT106b plasmid map

July 11

  

Wet Lab:

  • Recorded BL21 pDawn-mCherry growth curve and calculated doubling time
    • Miniprepped biobricks
    • BBa_K265008 - Ice Nucleation Protein NC
    • BBa_K523013 - Plac + INP-EYFP
    • BBa_K299810 - B0032 + Invasin
    • BBa_K257010 - ClyA + RFP
  

Dry Lab:

  • Worked on biofilm project schematic
  • More work on wiki

July 13

  

Wet Lab:

  • Diluted to OD600=0.01 and take 0-8hr time points of pDawn-mCherry fluorescence time course
  • Thawed out HEK293T cells
  

Dry Lab:

  • Worked on wiki/schematic

July 17

  

Wet Lab:

  • Re-ligation of pDawn with IDTClyA and ClyA-RFP, Transformation into DH5alpha/XO1blue
  • Performed PCR of plsr & gfp w/new primers.
  • Picked colonies of T7 and INPNC-DARPin
  

Dry Lab:

  • Designed experiments for ClyA and INPNC-DARPin assays

Week 7

July 16

  

Wet Lab:

  • Transformed INPNC-DARPin and T7 BioBrick
  

Dry Lab:

  • SAAST presentation
  • Met with Lazzara lab to discuss DARPin experiments

July 18

  

Wet Lab:

  • Re-inoculated T7 and INPNC-DARPin
  • PCR ClyA-RFP, Gel purify PCR ClyA-RFP product
  

Dry Lab:

  • Worked on DARPin assay experiment design

July 19

  

Wet Lab:

  • Trypan Blue Assay
  • Digested pDawn-mCherry with BamHI, NotI, pDawn-mCherry with BamHI,Digest pDawn-mCherry with NotI, Digested IDTsmart-ClyA with BamHI, NotI (Gel for Digests showed possible degeneration of NotI enzyme...)
  • Ligated pDawn backbone (old) with 1) ClyA and 2) ClyA-RFP and Ligated pDawn backbone (new) with ClyA and ClyA-RFP, as well as pDawn old backbone with H20 and pDawn new backbone with H20 (ligation controls)
  • Transformed all of above ligations into DH5alpha

July 23

  

Wet Lab:

  • Ashwin-Design test cuts for pDawn-Clya, pDawn-ClyA+RFP
  • Ashwin-Digest pDawn-ClyA+pDawn-ClyA+RFP, Run on gel
  • Nikita - Miniprep pDawn culture in Sarkar cold room
  • Digest of pET-26b w/ BglII, EcoRI-Peter
  • Digest of plsr product w/ BglII, PseI-Peter
  • Digest egfp w/ SpeI, EcoRI-Peter
  • Ligate & transform pET-26-plsr-GFP-Peter
  • Digest INPNC-DARPin
  • Avin - Try to rescue 293T cells after CO2 arrives if can’t be rescued, thaw out more 293T
  • Transform pET-26b-luxS - Peter
  • Avin - pick GAVPO
  

Dry Lab:

  • Ordered AI-2 ASAP-peter
  • Nikita - Researched whether ClyA is toxic to E. coli BL21, JT2, DH5 alpha, and whether it is secreted in these strains
  • Read Daphne+Dr. Sarkar’s ligation paper
    • Avin - Buy:
    • DH5 alpha
    • 20 kan plates, 10 amp plates
    • Cell Counter
  • Write out competent cell production protocol-Peter
  • Obtained BL21 transformation protocol from daphne/find it-Peter
  • Avin - Logo design, human practices
  • Ashwin - wiki

July 24

  

Wet Lab:

  • Pick colonies of pDawn-ClyA and pDawn-ClyA+RFP (DH5alpha)
  • Avin - Picked 2 colonies (light+dark) of pDawn-ClyA+RFP (BL21), set up pDawn culture experiment, check on HEK293T and passage into 6 well plates if confluent
  • Avin - Ran Gel, no DNA
  

Dry Lab:

  • Peter: order the promoter (or figure out an alternative method..)
  • Ashwin: wiki
  • Avin - Edit/Order primers for HA tag and His tag, design pDawn-ClyA experiments, human practices
  • Nikita - Write human practices

Week 8