iGEM 2012 — Team Penn

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# Team:Penn/Notebook

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Penn 2012 iGEM Wiki

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* [Our Project](/Team%3APenn/ProjectOverview)
  + [Motivation](/Team%3APenn/ProjectOverview)
  + [Our Therapeutic](/Team%3APenn/ProjectResults)
  + [Achievements](/Team%3APenn/Achievements)
* [Light Activation](/Team%3APenn/LightActivatedOverview)
  + [Overview](/Team%3APenn/LightActivatedOverview)
  + [YF1/FixJ BL Sensor](/Team%3APenn/BLSensor)
  + [Light Activated Cell Lysis](/Team%3APenn/LightActivatedLysis)
* [Surface Display](/Team%3APenn/SurfaceDisplayOverview)
  + [Overview](/Team%3APenn/SurfaceDisplayOverview)
  + [INPNC Surface Display](/Team%3APenn/SurfaceDisplay)
  + [HER2 Targeting](/Team%3APenn/Targeting)
  + [BBa Surface Display Platform](/Team%3APenn/SurfaceDisplayBBa)
* Wet Lab
  + [Notebook](/Team%3APenn/Notebook)
  + [Safety](/Team%3APenn/Safety)
  + [Protocols](/Team%3APenn/Protocols)
  + [Parts](/Team%3APenn/Parts)
* [Human Practices](/Team%3APenn/HumanPracticesOverview)
  + [Overview](/Team%3APenn/HumanPracticesOverview)
  + [Bacterial Therapeutics](/Team%3APenn/Barriers)
  + [Outreach](/Team%3APenn/Outreach)
  + [Public Perception](/Team%3APenn/Perceptions)
  + [E. Coli Nissle 1917](/Team%3APenn/Nissle)
  + [VerifiGEM](/Team%3APenn/VerifiGEM)
* [About Us](/Team%3APenn/Team)
  + [Team](/Team%3APenn/Team)
  + [Sponsors](/Team%3APenn/Sponsors)

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**

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

**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 2012 Notebook

## Week 9

**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

## Week 10

**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

## Week 11

**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

## Week 12

**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
* Also start cultures of 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

September 2012 Notebook

## Week 13

## Week 14

**September 10**

* Mike - pick INPNC-mCherry colony (transformed Sample #2) and split into + and - cultures
* Start BL21 cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA, His-Intein-mCherry
* Peter - Protein purification of His-DARPin-HA, ClyA-His
* Avin - Dilute (9am) & Induce (?) His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA at 0.5
* Ashwin - Wiki work

**September 11**

* Mike - Make glycerol stock of INPNC-mCherry, Dilute and induce INPNC-mCherry, INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin HA, His-Intein-mCherry
* Peter - Make electro/chemically competent cells, transform
* Ashwin - Pick up S. typhimurium from Mark Goulian

**September 12**

* Peter - Lysis and spin down (for INPNC-mCherry, aliquot 1mL of both (+) and (-) for Avin
* Peter- spin down and check if red, then lyse) , put in 4C, check Nissle eGFP colonies
* Avin - Immuno experiment on His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA, Take INPNC-mCherry, His-Intein-mCherry, fix and mount onto scope slides

**September 13**

* Mike - Bradford assay, Run protein gel
* Peter -
* Avin - Observe BL21 transformatons under confocal
* Ashwin - colony PCR on S. typhimurium for MisL

**September 14**

* Mike -
* Peter -
* Avin - core confocal?
* Ashwin -

Other

* Order scfv plasmid, scfv primers
* Order MisL Primers, pick up misL, PCR on S. typhimurium
* Chemical/Electrocompetent Nissle
* Make LB, Glycerol
* Wiki

## Week 15

## Week 16