Team:Penn/Notebook

From 2012.igem.org

(Difference between revisions)
 
(20 intermediate revisions not shown)
Line 1: Line 1:
-
<html><head>
+
{{:Team:Penn/Template/Site2}}
-
<link rel="stylesheet" href='/Team:Penn/css/navigation?action=raw&amp;ctype=text/css' />
+
 
-
<link rel="stylesheet" href='/Team:Penn/css/layerslider?action=raw&amp;ctype=text/css' />
+
<html>
-
<link rel="stylesheet" href='/Team:Penn/css/style2?action=raw&amp;ctype=text/css' />
+
<head>
-
<link rel="stylesheet" href='/Team:Penn/css/columns?action=raw&amp;ctype=text/css' />
+
-
<link rel="stylesheet" href='/Team:Penn/css/igemsubcss?action=raw&amp;ctype=text/css' />
+
-
<link rel="stylesheet" href='/Team:Penn/css/accordion?action=raw&amp;ctype=text/css' />
+
<script src='http://www.switchroyale.com/vallenato/js/jquery-1.5.min.js?action=raw&amp;ctype=text/javascript' type="text/javascript" charset="utf-8"></script>
<script src='http://www.switchroyale.com/vallenato/js/jquery-1.5.min.js?action=raw&amp;ctype=text/javascript' type="text/javascript" charset="utf-8"></script>
-
<script src='http://www.switchroyale.com/vallenato/vallenato/vallenato.js?action=raw&amp;ctype=text/javascript' type="text/javascript" charset="utf-8"></script>
+
<script src='https://2012.igem.org/Team:Penn/js/vallenato?action=raw&amp;ctype=text/javascript' type="text/javascript" charset="utf-8"></script>
<link rel="stylesheet" href='http://www.switchroyale.com/vallenato/vallenato/vallenato.css?action=raw&amp;ctype=text/css' type="text/css" media="screen" charset="utf-8">
<link rel="stylesheet" href='http://www.switchroyale.com/vallenato/vallenato/vallenato.css?action=raw&amp;ctype=text/css' type="text/css" media="screen" charset="utf-8">
-
<title>Penn 2012 iGEM Wiki</title>
 
-
<script type="text/javascript">
 
-
 
-
  var _gaq = _gaq || [];
 
-
  _gaq.push(['_setAccount', 'UA-5390553-2']);
 
-
  _gaq.push(['_trackPageview']);
 
-
 
-
  (function() {
 
-
    var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;
 
-
    ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';
 
-
    var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);
 
-
  })();
 
-
 
-
</script>
 
-
 
-
 
-
 
</head>
</head>
 +
<!----------------------------------------------------------------------------->
-
<body>
+
<p style="text-align:center;color:white;">June 2012 Notebook</p>
-
<div style="overflow:hidden;">
 
-
<div style="margin-bottom:0px; padding-bottom:0px;">
 
-
<a href="https://2012.igem.org/Team:Penn" style="padding:0px; margin:0px;">
 
-
<img src="https://static.igem.org/mediawiki/2012/e/e5/Bluelogo.jpg" /></a>
 
-
</div>
 
-
 
-
<ul class="nav">
 
-
<li class="dropdown">
 
-
 
-
    <a href="#">Drug Delivery</a>
 
-
 
-
<ul>
 
-
        <li><a href='/Team:Penn/DrugDeliveryOverview'>Overview</a></li>
 
-
<li><a href='/Team:Penn/DrugDeliverySystem'>System</a></li>
 
-
<li><a href='/Team:Penn/DrugDeliveryResults'>Results</a></li>
 
-
<li><a href='/Team:Penn/DrugDeliveryParts'>Parts</a></li>
 
-
</ul>
 
-
</li>
 
-
 
-
<li class="dropdown">
 
-
 
-
    <a href="#">Biofilms</a>
 
-
 
-
<ul>
 
-
              <li><a href='/Team:Penn/BiofilmsOverview'>Overview</a></li>
 
-
<li><a href='/Team:Penn/BioflmsSystem'>System</a></li>
 
-
<li><a href='/Team:Penn/BiofilmsResults'>Results</a></li>
 
-
<li><a href='/Team:Penn/BiofilmsParts'>Parts</a></li>
 
-
</ul>
 
-
</li>
 
-
 
-
<li class="dropdown">
 
-
 
-
<a href="#">Lab Work</a>
 
-
 
-
<ul>
 
-
 
-
                                <li><a href='/Team:Penn/Notebook'>Notebook</a></li>
 
-
<li><a href='/Team:Penn/Safety'>Safety</a></li>
 
-
<li><a href='/Team:Penn/Protocols'>Protocols</a></li>
 
-
 
-
</ul>
 
-
 
-
        </li>
 
-
 
-
<li class="dropdown">
 
-
 
-
<a href="#">Human Practices</a>
 
-
 
-
<ul>
 
-
 
-
<li><a href='/Team:Penn/Education'>Education</a></li>
 
-
<li><a href='/Team:Penn/Regulations'>Regulations</a></li>
 
-
<li><a href='/Team:Penn/Registry'>The Registry</a></li>
 
-
</ul>
 
-
 
-
</li>
 
-
 
-
<li class="dropdown">
 
-
 
-
<a href="#">About Us</a>
 
-
 
-
<ul>
 
-
 
-
<li><a href='/Team:Penn/Team'>Team</a></li>
 
-
<li><a href='/Team:Penn/Advisors'>Advisors</a></li>
 
-
<li><a href='/Team:Penn/Sponsors'>Sponsors</a></li>
 
-
</ul>
 
-
</li>
 
-
 
-
<div class="arrow"></div>
 
-
 
-
</ul>
 
-
 
-
<!----------------------------------------------------------------------------->
 
-
<p style="text-align:center">June 2012 Notebook</p>
 
<div id="accordion-container">
<div id="accordion-container">
<h2 class="accordion-header">Week 1</h2>
<h2 class="accordion-header">Week 1</h2>
Line 117: Line 22:
                                                   <li>Set up some lab equipment</li>
                                                   <li>Set up some lab equipment</li>
                                                   <li>Autoclaved for a while</li>
                                                   <li>Autoclaved for a while</li>
-
<li>Organized biobrick stuff</li>
+
                                                  <li>Organized biobrick stuff</li>
                                                   <li>Called Vinoo about DNA planning</li>
                                                   <li>Called Vinoo about DNA planning</li>
    
    
Line 139: Line 44:
<p><b>June 11th</b></p>
<p><b>June 11th</b></p>
-
                             &nbsp;&nbsp;<p>Wet Lab</p>
+
                             &nbsp;<p>Wet Lab</p>
                                                 <ul>
                                                 <ul>
                                                   <li>PCR'd mCherry from NAS157</li>
                                                   <li>PCR'd mCherry from NAS157</li>
Line 152: Line 57:
<br>
<br>
<p><b>June 12th</b></p>
<p><b>June 12th</b></p>
-
                             &nbsp;&nbsp;<p>Wet Lab</p>
+
                             &nbsp;<p>Wet Lab</p>
                                                 <ul>
                                                 <ul>
                                                   <li>Digested mCherry PCR product with BamHI and NotI</li>
                                                   <li>Digested mCherry PCR product with BamHI and NotI</li>
Line 166: Line 71:
<br>
<br>
<p><b>June 14th</b></p>
<p><b>June 14th</b></p>
-
&nbsp;&nbsp;<p>Wet Lab</p>
+
 
-
                                                <ul>
+
-
                                                  <li>Fill in later....</li>
+
-
                                               
+
-
                                                </ul>
+
&nbsp;&nbsp;<p>Dry Lab</p>
&nbsp;&nbsp;<p>Dry Lab</p>
<ul>
<ul>
Line 308: Line 209:
</div>
</div>
</div>
</div>
-
<p style="text-align:center">July 2012 Notebook</p>
+
<p style="text-align:center; color:white;">July 2012 Notebook</p>
<div id="accordion-container">
<div id="accordion-container">
<h2 class="accordion-header">Week 5</h2>
<h2 class="accordion-header">Week 5</h2>
Line 716: Line 617:
</div>
</div>
-
<p style="text-align:center">August 2012 Notebook</p>
+
<p style="text-align:center;color:white;">August 2012 Notebook</p>
<div id="accordion-container">
<div id="accordion-container">
<h2 class="accordion-header">Week 9</h2>
<h2 class="accordion-header">Week 9</h2>
Line 1,132: Line 1,033:
</ul>
</ul>
<br>
<br>
-
                        </div>
 
 +
<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</li><li> Also start cultures of pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>August 29</b></p>
 +
<ul>
 +
<li>
 +
Induce His-DARPin-HA culture (100 microliters of 1 M IPTG)</li>
 +
<li>
 +
Monitor SK-BR-3 </li>
 +
<li>
 +
Primers for ClyA into lactococcus</li>
 +
<li>
 +
Primers for INPNC-mCherry-DARPin </li>
 +
<li>
 +
Pick colonies for pBAD33-eGFP if the RT ligation and transformation worked - Peter/Ashwin</li>
 +
<li>
 +
If RT ligation and transformation looks bad, transform the 4C and 16C ligations - Mike</li>
 +
<li>
 +
When primers arrive, PCR out mCherry from JMIL intein plasmid, PCR out INPNC, then assembly PCR for INPNC-mCherry</li>
 +
</ul>
 +
<br>
 +
 +
 +
<p><b>August 30</b></p>
 +
<ul>
 +
<li>
 +
Protein purification of His-DARPin-HA
 +
</li>
 +
<li>
 +
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
 +
</li>
 +
<li>
 +
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
 +
</li>
 +
<li>
 +
Start DH5a cultures for INPNC, INPNC-HA, His-DARPin-HA, pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
 +
</li>
 +
<li>
 +
Repick colonies of pBAD33-eGFP from 16C or 4C plate
 +
</li>
 +
</ul>
 +
<br>
 +
                   
 +
  </div>
</div>
</div>
Line 1,139: Line 1,125:
 +
<p style="text-align:center;color:white;">September 2012 Notebook</p>
 +
<div id="accordion-container">
 +
<h2 class="accordion-header">Week 13</h2>
 +
<div class="accordion-content">
 +
                        </div>
 +
<h2 class="accordion-header">Week 14</h2>
 +
 +
<div class="accordion-content">
 +
<p><b>September 10</b></p>
 +
<ul>
 +
<li>
 +
Mike - pick INPNC-mCherry colony (transformed Sample #2) and split into + and - cultures
 +
</li>
 +
<li>Start BL21 cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA, His-Intein-mCherry
 +
</li>
 +
<li>
 +
Peter - Protein purification of His-DARPin-HA, ClyA-His
 +
</li>
 +
<li>
 +
Avin - Dilute (9am) & Induce (?) His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA at 0.5
 +
</li>
 +
<li>
 +
Ashwin - Wiki work
 +
</li>
 +
</ul>
 +
<br>
 +
<p><b>September 11</b></p>
 +
<ul>
 +
<li>
 +
Mike - Make glycerol stock of INPNC-mCherry, Dilute and induce INPNC-mCherry, INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin HA, His-Intein-mCherry
 +
</li>
 +
<li>
 +
Peter - Make electro/chemically competent cells, transform
 +
</li>
 +
<li>
 +
Ashwin - Pick up S. typhimurium from Mark Goulian
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>September 12</b></p>
 +
<ul>
 +
<li>
 +
Peter - Lysis and spin down (for INPNC-mCherry, aliquot  1mL of both (+) and (-) for Avin
 +
</li>
 +
<li>Peter- spin down and check if red, then lyse) , put in 4C, check Nissle eGFP colonies
 +
</li>
 +
<li>
 +
Avin - Immuno experiment on His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA, Take INPNC-mCherry, His-Intein-mCherry, fix and mount onto scope slides
 +
</li>
 +
</ul>
 +
</br>
 +
 +
<p><b>September 13</b></p>
 +
<ul>
 +
<li>
 +
Mike - Bradford assay, Run protein gel</li>
 +
<li>Peter - </li>
 +
<li>
 +
Avin - Observe BL21 transformatons under confocal
 +
</li>
 +
<li>
 +
Ashwin - colony PCR on S. typhimurium for MisL
 +
</li>
 +
</ul>
 +
<br>
 +
 +
<p><b>September 14</b></p>
 +
<ul>
 +
<li>
 +
Mike -
 +
</li>
 +
<li>
 +
Peter -
 +
</li>
 +
<li>
 +
Avin - core confocal?
 +
</li>
 +
<li>
 +
Ashwin -
 +
</li>
 +
</ul>
 +
 +
&nbsp;&nbsp;<p>Other<p>
 +
<ul>
 +
<li>Order scfv plasmid, scfv primers</li>
 +
<li>
 +
Order MisL Primers, pick up misL, PCR on S. typhimurium
 +
</li>
 +
<li>
 +
Chemical/Electrocompetent Nissle
 +
</li>
 +
<li>
 +
Make LB, Glycerol
 +
</li>
 +
<li>
 +
Wiki
 +
</li>
 +
</ul>
 +
</br>
 +
                        </div>
 +
 +
<h2 class="accordion-header">Week 15</h2>
 +
 +
<div class="accordion-content">
 +
 +
                        </div>
 +
 +
<h2 class="accordion-header">Week 16</h2>
 +
 +
<div class="accordion-content">
 +
 +
                        </div>
 +
 +
 +
</div>

Latest revision as of 05:52, 21 October 2012

Penn 2012 iGEM Wiki

Image Map

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

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