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

July 26

  

Wet Lab:

• Avin - Finish ClyA-RFP time course, collect ClyA supernatant, measure fluorescence of ClyA-RFP triplicates, spin down + pictures if red, Miniprep of DARPin, possible ClyA assay
• Peter- ligation, transformation, plating of ligations, help Avin with ClyA cell lysis experiments
• Transform luxS6-8 into BL21
• Nikita - Miniprep GAVPO and DARPin

  

Dry Lab:

• Avin - Call IDT at 9am, order blood agar plates, nissl stuff after we do background research
• Ashwin - wiki, research getting recombinant ClyA
• Nikita - Human practices, Nissle research, plan experiments
• Peter
  • Complete FDA writeup
  • Order ATCC Strains
  • Order lss primers
  • Order AI-2
  • Order Crystal Violet Stain
  • Plan ClyA experiment in JMOutline

July 30

  

Wet Lab:

• Avin - Transform pBAD33 and pSB4A5, Inoculate BL21 Intein colonies
• Redo the test cuts for pDawn and pDawn-ClyA-RFP with XmaI and ClaI on a 0.7% gel

  

Dry Lab:

• Avin - Human Practices
• Mike - Primers
  • pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse
  • pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, figure out reverse
  • pet26b-ClyA+RFP keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse
  • pet26B-CLlyA+RFP removing pelB with 6x His C terminus - use NdeI on forward primer, figure out reverse
  • fix frame shift in pDawn and include 6xHis N terminus - use NdeI on forward primer, figure out reverse
  • ClyA
  • mCherry
  • ClyA-RFP
  • Cph8 primers - figure out pJT106b primers

July 31

  

Wet Lab:

• Test Cut pDawn-ClyA-RFP colony 4 w/ XmaI and Cla
• Transform the pDawn-ClyA-RFP colony 4 plasmid (assuming test cuts look good) into BL21 and Dh5alpha
• Check for growth of Intein-mCherry plasmid
• Assuming growth:
  • miniprep DH5alpha culture
  • induce BL21 culture with IPTG - see Jordan for protocol
  • If primers arrive → PCR (x2) for DARPin (check order status for IDT in the morning, call cell center if it says its been shipped)
  • Pick colonies and innoculate pBAD33 (chloramphenicol) and pSB4A5 (ampicillin)

  

Dry Lab:

• Mike - Order Primers
  • pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, XhoI on reverse (no stop codon)
  • pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, XhoI on reverse (no stop codon)
  • fix frame shift in pET26b for ClyA-RFP (NdeI on forward, NotI on reverse, include stop) - can’t use NdeI because there is an NdeI cut site inside ClyA-RFP
  • fix frame shift in pDawn and include 6xHis N terminus
  • ClyA - NdeI on forward, BamHI on reverse
  • mCherry - NdeI on forward, NotI on reverse
  • ClyA-RFP - NdeI on forward, NotI on reverse - can’t use NdeI because there’s an NdeI cut site inside ClyA-RFP
  • Cph8 primers - figure out pJT106b primers
• Mike - Take out Biohazard for Sevile
• Ashwin - wiki

August 1st

  

Wet Lab:

pET-26-plsr-gfp triple ligation ( (+) indicates positive control, (-) indicates negative control)

Gel Purification 8:30-12:00
• 1% gel 50mL +5uL SyberSafe
• pET-26b cut w/ EcoRI-HF (+)
• pET-26b cut w/ EcoRI-HF & BglII
• run against pET-26b uncut for reference

Ligation (Start w/ 25ng vector) 12:00-3:00
• Vector:GFP:plsr
• 1:6:6
• 1:4:4
• 1:1:1
• pET-26b cut w/ EcoRI-HF (+)
• pET-26b cut w/ EcoRI-HF & BglII (-)
• INCUBATE @ RT 1hr
• Transform Into DH5a Max Efficiency 3:00-6:00
  • Transform pET-26b (+)
  • Transform H2O (-)
  • Transform 1:6:6
  • Transform 1:4:4
  • Transform 1:1:1
• Total Plates Needed=8
• Check IDT primer order in the morning
• Avin - Pick colonies of pDawn-ClyA-RFP (DH5a and BL21) and inoculate into 5 mL LB culture
• Avin - Miniprep pSB4A5, pBAD33, Intein-mCherry (DH5a) Grow up BL21 Intein-mCherry and induce for fun?

August 2

  

Wet Lab:

• pDawn-ClyA-RFP Time course - Avin
• growth to 0.8 and 1:1000 IPTG induction of 50mL BL21 Intein-mCherry - Avin
• Miniprep of DH5 alpha pDawn-ClyA-RFP - Ashwin
• PCR purify INPNC-DARPin-HA Tag - Mike
• Digest INPNC-DARPin-HA Tag and pET26b with EcoRI and NdeI
• Column purify INPNC-DARPin-HA Tag
• Gel purify pET26b
• Digest INPNC, INPNC-HA Tag, 6xHis-DARPin-Ha Tag with NdeI and BamHI
• Column purify INPNC, INPNC-HA Tag, 6xHis-DARPin-HA Tag
• Gel purify PET26b
• Gel purification of pET-26b digested with BglII and EcoRI-HF
• Plated S. Epidermis and E. Coli containing Lysostaphin

  

Dry Lab:

• Logo - Avin

August 3

  

Wet Lab:

• BL21 pDawn-ClyA-RFP time course, store blood agar plates in fridge - Avin
• BL21 Intein-mCherry Protein purification and Coomassie gel - Avin/Peter
• Ligate and transform pET26b-INPNC, pET26b-INPNC-HATag, pET26b-INPNC-DARPin-HATag, pET26b-6xHis-DARPin-HATag
• Saturday - pick colonies
• Sunday/Monday - Miniprep, test cuts, transformation
• Check if ClyA primers are shipped, if so pick up at cell center
• PCR ClyA with various primers
• Run on Gel, Gel Purify, Digest, Column Purify, Ligate, Transform

  

Dry Lab:

• Pick up primers
• Meeting with Dr. Sarkar

August 13

• Started pDawn ClyA & pDawn ClyA-RFP cultures for blood agar experiment
• Meeting with Dr. Sarkar to discuss progress on both projects

August 14

• Reviewed sequencing results and transformed the following into BL21 and DH5 alpha:
  • pET26b-INPNC
  • pET26b-INPNC-HA
  • pET26b-6xHis-DARPin-HA
  • pET26b-INPNC-DARPin-HA
  • pET26b-ClyA-6xHis
  • pET26b-PelB-ClyA-6xHis
  • pDawn-ClyA-6xHis
• Serial dilution and plating of pDawn ClyA & pDawn ClyA-RFP cultures for blood agar experiment

August 15

• Re-plated all constructs
• pDawn-ClyA and pDawn-ClyA-RFP demonstrated light-dependent hemolysis!
• BL21 washing titration experiment for flow cytometry experiment - obtained optimal starting OD600 of 0.05 to obtain ~1E6 cells after all washes and incubations
• Made Incubation Buffer for flow cytometry experiment
• Designed/ordered pBAD33-eGFP Primers

August 16

• Picked colonies on all BL21 and DH5 constructs, including inducing and non-inducing conditions, set up flow cytometry stuff, booked flow core
• Started cultures of pDawn-ClyA, pDawn-ClyA-RFP, and pDawn-ClyA-mCherry for repeat blood agar experiment
• Set up light incubator properly
• Started protein purification cultures of pDawn-ClyA-6xHis, pET26b-ClyA-6xHis, and pET26b-PelB-ClyA-6xHis, grew to OD600=0.8, induced with IPTG or light

August 22

• Image INPNC-DARPin-HA, INPNC-HA, DARPin-HA (both induced and not induced) on the confocal - Avin
• Figure out ClyA imaging system and maybe design a stencil (not a high priority)
• Pick up media and everything else from cell center - Mike
• Ligate ClyA into lactococcus? -- Talk to Daphne about vectors
• Order the cytotoxicity assay from Promega - Avin
• Ask Dr. Sarkar for cell center account - Mike
• Order pDawn sequencing primer - Avin
• Run GFP PCR product on Gel, Gel didn’t work → rerun PCR using gradient annealing temp Run PCR products on gel, check if works
• Look into biobrick format - Mike
• Make an in-depth plan for the incoming weeks, prioritizing what needs to be done Cloning! what is the final construct? Design and execute add RBS to pBAD33 primer

Plan for August 23

• Add RBS/SD to pBAD33 forward primer and re-order it
• Send out sequencing for INPNC-DARPin-HA (and what else?)
• Look at old sequencing data just to make sure everything worked
• Pick up IPTG from the cell center and make stock solution/aliquots
• Using new IPTG, spread on blood agar plates (final dilution of 1 to 1000)
• Spread Kan on Blood Agar Plates
• Plate pET26b-ClyA-His (+/-), pET26b-pelB-ClyA-His (+/-), and pDawn-ClyA(FS) (+/-) on Blood Agar + Kan plates

August 24

• If colonies were picked, miniprep, then test cut, then run on diagnostic gel → transform into BL21
• If colonies were not picked, then pick colonies
• Analyze sequencing results
• Plan out INPNC-DARPin-HA experiments
• Figure out cloning for 1 plasmid system

Goals in order of priority:

• Show that INPNC-DARPin-HA binds to HER2 in vitro
• Show that purified 6xHis-DARPin-HA binds to Her2 - Monday?
• Show that INPNC-DARPin-HA is displayed on the surface
• Show INPNC-mCherry is displayed on surface (confocal?)
• Construct ClyA and ClyA-His BioBricks
• Construct INPNC-mCherry Biobrick
• Construct Surface Display BioBrick (need INPNC-mCherry to work)
• Transform ClyA into Lactococcus and plate onto blood agar for Human Practices-
• Stencil experiment showing spatial control of pDawn-ClyA (could be done next week, only takes a little time and artistic ability)
• Show that cph8 works with a reporter
• Show that cph8-ClyA works

Experiments in order of priority:

• Construction of pBAD33-eGFP (Mike)
• Order primers for ClyA-His and ClyA to clone them into BioBrick backbone
• Order primers for INPNC-mCherry
• Order primers for cph8-reporter, cph8-clyA-his
• SKBR3 imaging with pBAD33-eGFP/pET26b-INPNC-DARPin-HA bacteria on confocal
• INPNC-DARPin immunos, 0.2mM IPTG, overnight induction
• INPNC-DARPin bacterial flow cytometry on FACS machine (can use same bacteria as immuno)
• Create stable cell line of SKBR3-mCherry (Jordan)
• Obtain Lactococcus expression vector, order primers to clone in ClyA-His
• Design surface display biobrick
• Cytotoxicity experiment on SKBR3
• Protein purification on His-DARPin-HA (Peter)
• Flow cytometry of SKBR3 cells incubated with his-DARPin-HA (someone/Najaf)
• Construct cph8-ClyA plasmid
• cph8-reporter timecourse
• cph8-ClyA blood agar

August 27

• PCR eGFP out of PHAT (annealing = 65), run on 1% gel, gel purify
• Digest eGFP and pBAD33 with PstI and XmaI, column purify
• Ligate at RT for 1 hour, transform into DH5a depending on time, otherwise ligate at 16C/4C overnight and transform the next day
• Order Primers for (in order of priority)
• ClyA and ClyA-His Biobricks
• INPNC-mCherry construct
• ClyA into lactococcus plasmid
• Cph8-reporter plasmid
• Cph8-ClyA-His plasmid
• INPNC surface display vector (provided it works)
• Start cell culture of SK-BR-3 cells?
• Get protocols

August 28

• start cultures for purification of His-DARPin-HA
• HA affinity purification of INPNC-DARPin-HA?
• Print and hand in safety form to EHRS
• Pick up SK-BR-3 plate from Cal, learn protocols
• Add FBS to our McCoy’s media
• Design and order primers to put ClyA into lactococcus expression vector
• Set up preliminary stencil experiment, send avin plate base diameter (approximate 86mm)
• Design INPNC-mCherry-DARPin primers
• pBAD33-eGFP
• Run Gradient PCR products (which ran overnight) on gel, gel purify the best band
• Digest eGFP and pBAD33 with PstI and XmaI, column purify/gel purify
• Ligate at RT for 1 hour, transform into DH5a depending on time, otherwise ligate at 16C/4C overnight and transform the next day
• Start DH5 alpha cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA, pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps

August 29

• Induce His-DARPin-HA culture (100 microliters of 1 M IPTG)
• Monitor SK-BR-3
• Primers for ClyA into lactococcus
• Primers for INPNC-mCherry-DARPin
• Pick colonies for pBAD33-eGFP if the RT ligation and transformation worked - Peter/Ashwin
• If RT ligation and transformation looks bad, transform the 4C and 16C ligations - Mike
• When primers arrive, PCR out mCherry from JMIL intein plasmid, PCR out INPNC, then assembly PCR for INPNC-mCherry

August 30

• Protein purification of His-DARPin-HA
• PCR purify INPNC-mCherry, run a few ul on a diagnostic gel → results call for gel purifiation → Gel purify INPNC-mCherry → yield was bad, redo PCR → gel purify
• Digest INPNC-mCherry and pET26b with NdeI and HindIII, ligate at RT for 1 hr (and maybe some at 4C or 16C overnight), and transform into pET26b
• Start DH5a cultures for INPNC, INPNC-HA, His-DARPin-HA, pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
• Repick colonies of pBAD33-eGFP from 16C or 4C plate