Team:Penn/Notebook

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

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

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)

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 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

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

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