Team:Penn/Notebook
From 2012.igem.org
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Get protocols | Get protocols | ||
+ | </li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <p><b>August 28</b></p> | ||
+ | <ul> | ||
+ | <li> | ||
+ | start cultures for purification of His-DARPin-HA | ||
+ | </li> | ||
+ | <li> | ||
+ | HA affinity purification of INPNC-DARPin-HA? | ||
+ | </li> | ||
+ | <li> | ||
+ | Print and hand in safety form to EHRS | ||
+ | </li> | ||
+ | <li> | ||
+ | Pick up SK-BR-3 plate from Cal, learn protocols | ||
+ | </li> | ||
+ | <li> | ||
+ | Add FBS to our McCoy’s media | ||
+ | </li> | ||
+ | <li> | ||
+ | Design and order primers to put ClyA into lactococcus expression vector | ||
+ | </li> | ||
+ | <li> | ||
+ | Set up preliminary stencil experiment, send avin plate base diameter (approximate 86mm) | ||
+ | </li> | ||
+ | <li> | ||
+ | Design INPNC-mCherry-DARPin primers | ||
+ | </li> | ||
+ | <li> | ||
+ | pBAD33-eGFP | ||
+ | </li> | ||
+ | <li> | ||
+ | Run Gradient PCR products (which ran overnight) on gel, gel purify the best band | ||
+ | </li> | ||
+ | <li> | ||
+ | Digest eGFP and pBAD33 with PstI and XmaI, column purify/gel purify | ||
+ | </li> | ||
+ | <li> | ||
+ | Ligate at RT for 1 hour, transform into DH5a | ||
+ | depending on time, otherwise ligate at 16C/4C overnight and transform the next day | ||
+ | </li> | ||
+ | <li> | ||
+ | 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 | ||
</li> | </li> | ||
</ul> | </ul> |
Revision as of 13:05, 15 September 2012
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
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
August 2012 Notebook
Week 9
August 1st
Wet Lab:
-
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, pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps