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:
Order AI-2 ASAP-peter
Nikita - Research 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
Get BL21 transformation protocol from daphne/find it-Peter
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