Team:Penn/Notebook

From 2012.igem.org

(Difference between revisions)
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</ul>
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<p><b>July 31</b></p>
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&nbsp;&nbsp;<p>Wet Lab:</p>
 +
<ul>
 +
<li>
 +
Test Cut pDawn-ClyA-RFP colony 4 w/ XmaI and Cla</li>
 +
<li>
 +
Transform the pDawn-ClyA-RFP colony 4 plasmid (assuming test cuts look good) into BL21 and Dh5alpha</li>
 +
<li>
 +
Check for growth of Intein-mCherry plasmid</li>
 +
<li><ul>
 +
Assuming growth:
 +
<li>miniprep DH5alpha culture</li>
 +
<li>induce BL21 culture with IPTG - see Jordan for protocol</li>
 +
<dilute in morning, when it gets to 0.8, induce with IPTG (what concentration) and then in a few hours (2-4), can spin down and see pellet</li>
 +
<li>If primers arrive → PCR (x2) for DARPin (check order status for IDT in the morning, call cell center if it says its been shipped)</li>
 +
<li>Pick colonies and innoculate pBAD33 (chloramphenicol) and pSB4A5 (ampicillin)</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
 +
&nbsp;&nbsp;<p>Dry Lab:</p>
 +
<ul>
 +
<li>Mike - Order Primers<ul></li>
 +
<li>
 +
pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, XhoI on reverse (no stop codon)</li>
 +
<li>
 +
pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, XhoI on reverse (no stop codon)
 +
</li>
 +
<li>
 +
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
 +
</li>
 +
<li>
 +
fix frame shift in pDawn and include 6xHis N terminus
 +
</li>
 +
<li>
 +
ClyA - NdeI on forward, BamHI on reverse
 +
</li>
 +
<li>
 +
mCherry - NdeI on forward, NotI on reverse
 +
</li>
 +
<li>
 +
ClyA-RFP - NdeI on forward, NotI on reverse - can’t use NdeI because there’s an NdeI cut site inside ClyA-RFP
 +
</li>
 +
<li>
 +
Cph8 primers - figure out pJT106b primers
 +
</li>
 +
<li>
 +
Mike - Take out Biohazard for Sevile
 +
</li>
 +
<li>
 +
Ashwin - wiki
 +
</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
<br>

Revision as of 12:05, 15 September 2012

Penn 2012 iGEM Wiki

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
    • Mike - Take out Biohazard for Sevile
    • Ashwin - wiki

Retrieved from "http://2012.igem.org/Team:Penn/Notebook"