Difference between revisions of "Team:Goettingen/Notebook"

 
(13 intermediate revisions by 2 users not shown)
Line 15: Line 15:
 
         padding:5px;
 
         padding:5px;
 
         line-height: 1.5;
 
         line-height: 1.5;
−
    }
+
            transition: background 0.8s;
 +
            -moz-transition: background 0.8s;
 +
            -webkit-transition: background 0.8s;
 +
            -o-transition: background 0.8s;
 +
            -ms-transition: background 0.8s;
 +
        }
 +
 
 +
        h2:hover {
 +
            color:white;
 +
            background-color:#7fbbe4;
 +
            transition: background 0.8s;
 +
            -moz-transition: background 0.8s;
 +
            -webkit-transition: background 0.8s;
 +
            -o-transition: background 0.8s;
 +
            -ms-transition: background 0.8s;
 +
        }
  
 
     h2 a {
 
     h2 a {
Line 23: Line 38:
 
     }
 
     }
  
−
     #menu1, #menu2, #menu3, #menu4, #menu5, #menu6, #menu7, #menu8, #menu9, #menu10, #menu11, #menu12{
+
     #menu1, #menu2, #menu3, #menu4, #menu5, #menu6, #menu7, #menu8, #menu9, #menu10, #menu11, #menu12, #menu13, #menu14, #menu15, #menu16, #menu17, #menu18, #menu19, #menu20, #menu21, #menu22, #menu23, #menu24, #menu25{
 
         display:none;
 
         display:none;
 
         width:90%;
 
         width:90%;
Line 44: Line 59:
 
<p>
 
<p>
 
<h1> Notebook </h1>
 
<h1> Notebook </h1>
 +
 
</p>
 
</p>
  
Line 801: Line 817:
 
</div>
 
</div>
  
−
<a href="" onClick=" $('#menu11').slideToggle(300, function callback() {  }); return false;"><h2> 23.3.15</h2></a>
+
<a href="" onClick=" $('#menu11').slideToggle(300, function callback() {  }); return false;"><h2> 23.03.15</h2></a>
  
 
     <div id="menu11">
 
     <div id="menu11">
Line 913: Line 929:
 
</p>
 
</p>
 
</div>
 
</div>
 +
 +
<a href="" onClick=" $('#menu23').slideToggle(300, function callback() {  }); return false;"><h2>13.04.15</h2></a>
 +
 +
<div id="menu23">
 +
<p>
 +
<p>
 +
    <strong>1.) Website:</strong>
 +
</p>
 +
<p>
 +
    Individual skills need to be worked out to find out who can do which part. Joschy is currently learning how to use HTML. He has made a navigation bar. It
 +
    may be helpful to have some external input.
 +
</p>
 +
<p>
 +
    The colour scheme will be our iGEM colours (blues)
 +
</p>
 +
<p>
 +
    The content must be updateable by every group, can be taken from the booklet to start with
 +
</p>
 +
<p>
 +
    Every team member needs to write a max. 150 word paragraph about themselves and submit a photograph to the dropbox.
 +
</p>
 +
<p>
 +
    <strong>2.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Dockerins: DNA has been extracted and we may get 3-4 Dockerins. Dockerins need to be finished in order to have the enzyme groups continue.
 +
</p>
 +
<p>
 +
    Scaffoldin: Transformation into E.coli worked and the plasmids can now be extracted and verified. First expression tests can follow. Alaa and Sindhu will
 +
    continue with the Scaffoldin from tomorrow.
 +
</p>
 +
<p>
 +
    E2: Primers will be designed on Thursday
 +
</p>
 +
<p>
 +
    E1: nothing to report due to a Practical, no work can be done atm.
 +
</p>
 +
<p>
 +
    Clone Manager: Julian will try to see if we can get access to it somehow.
 +
</p>
 +
<p>
 +
    <strong>3.) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    GZMB is meeting soon and will decide on funding. We have so far got 4 Sponsors.
 +
</p>
 +
<p>
 +
    <strong>4.) Flights:</strong>
 +
</p>
 +
<p>
 +
    Alaa has looked up flights if we book now:
 +
</p>
 +
<p>
 +
    700-870€, Hotel: 45-60€ p.P /pN. We need to book fast as most hotels and hostels will be booked by fellow igemers.
 +
</p>
 +
<p>
 +
    <strong>5.) Costs:</strong>
 +
</p>
 +
<p>
 +
    Anticipated Labcosts: 18000€
 +
</p>
 +
<p>
 +
    Flights: 800€ pP
 +
</p>
 +
<p>
 +
    Hotel: 600€ pP
 +
</p>
 +
<p>
 +
    We need a minimum of 6000€ from the GZMB if we want to continue with the project.
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu24').slideToggle(300, function callback() {  }); return false;"><h2>21.05.15 </h2></a>
 +
 +
<div id="menu24">
 +
<p>
 +
<p>
 +
    About the supervisors needs to be written for our Webpage
 +
</p>
 +
<p>
 +
    <strong>Labwork:</strong>
 +
</p>
 +
<p>
 +
    Dockerins: Attempt to put them in pBAD has failed so far. Retransformation control worked however. ACEL needs to be reinserted into pJET, BCEL needs to be
 +
    restarted from PCR, CTHE ligation needs to be redone. Change elution steps to gain higher concentrations (30µl instead of 50)
 +
</p>
 +
</div>
 +
 +
 +
<a href="" onClick=" $('#menu13').slideToggle(300, function callback() {  }); return false;"><h2> 19.06.15</h2></a>
 +
 +
    <div id="menu13">
 +
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Heiko will have an iGEM meeting slot everyday from 9-9.30am in his office.
 +
</p>
 +
<p>
 +
    E1:
 +
</p>
 +
<p>
 +
    -skipping BL21 step, because Esterase showed activity in TOP10 activity plates.
 +
</p>
 +
<p>
 +
    - Another option is to add a T7 Plasmid to induce the gene
 +
</p>
 +
<p>
 +
    - Phosphatase is expressing nicely (blue)
 +
</p>
 +
<p>
 +
    Phosphatase will be sent for sequencing next week.
 +
</p>
 +
<p>
 +
    E3:
 +
</p>
 +
<p>
 +
    Ready for expression plates with Cellulase in BL21
 +
</p>
 +
<p>
 +
    Scaffoldin:
 +
</p>
 +
<p>
 +
    Has not worked yet, probably due to the unspecific binding of the primers (good binding conditions are hard to find). Using a ligase free ligation in
 +
    pet101 which is sensitive to temperature and ions
 +
</p>
 +
<p>
 +
    -&gt;do a microdialysis to clean ligation from salts and ions before doing transformation.
 +
</p>
 +
<p>
 +
    The second scaffoldin was ordered and should arrive in the next couple of weeks.
 +
</p>
 +
<p>
 +
    RFP:
 +
</p>
 +
<p>
 +
    Cells are not pink and don’t fluoresce under the fluorescence microscope. Sequences are fine but will be done again in order to make sure.
 +
</p>
 +
 +
</div>
 +
 +
<a href="" onClick=" $('#menu14').slideToggle(300, function callback() {  }); return false;"><h2> 25.06.15</h2></a>
 +
 +
    <div id="menu14">
 +
 +
<p>
 +
    Present: Heiko, Ela, Julian, Dennis, Steffi, Avi, Verena, Alaa, Debbie, Nida
 +
</p>
 +
<p>
 +
    <strong>1.) Strain documentation: </strong>
 +
</p>
 +
<p>
 +
    The team has now got a box in the -80 ̊C freezer to start a strain collection. The Box is on the ground floor, in the middle freezer, top compartment, top
 +
    shelf marked: Rack 1 Robert at the back, 2<sup>nd</sup> from the bottom (blue box)
 +
</p>
 +
<p>
 +
    - To register the strain an information sheet needs to be filled in and submitted to Jaqueline
 +
</p>
 +
<p>
 +
    - Strains should be frozen from overnight culture in 30% glycerine (e.g. 600ul liquid culture + 900ul Glycerine (from 50% stock solution))
 +
</p>
 +
<p>
 +
    <strong>2.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Cellulase:
 +
</p>
 +
<p>
 +
    pJET_cellulase transformation and restriction worked fine in TOP10 and was then transformed into BL21 cells. Restriction showed a fragment of the correct
 +
    size, however sequencing failed. Activity plates showed no activity so far, but will be incubated over a longer period of time and in addition singularised
 +
    to avoid contamination of other colonies.
 +
</p>
 +
<p>
 +
    Scaffoldin:
 +
</p>
 +
<p>
 +
    Trafo in Top10 and BL21 did not work, which is why the TOPO cloning may be problematic.
 +
</p>
 +
<p>
 +
    - A control with an empty vector should be run.
 +
</p>
 +
<p>
 +
    - If the annealing is successful the bands should change in a reaction, which can be verified to exclude those issues.
 +
</p>
 +
<p>
 +
    - In addition Primers should be designed from within the sequence to verify the ends of the scaffoldin. Primers should be around 18bp long and 200bp within
 +
    the construct, which also has TOPO overlaps
 +
</p>
 +
<p>
 +
    - In addition the scaffoldin band of the PCR could be correct, but with an impurity alongside -&gt; transformation should however still work to some extent
 +
    (second problem?)
 +
</p>
 +
<p>
 +
    - For the covalent binding stick to the protocol an do not reduce salt concentrations
 +
</p>
 +
<p>
 +
    - For electroporation the TOPO cloning mixture needs to be desalinated with microdialysis, otherwise the trafo won't work
 +
</p>
 +
<p>
 +
    Recommendation: Check salts, positive control, annealing conditions and sequencing after PCR purification.
 +
</p>
 +
<p>
 +
    RFP:
 +
</p>
 +
<p>
 +
    Sequences are 100%match, but colonies show no fluorescence. Therefore, restricted RFP sequences will be ligated into pBAD in order to verify its function
 +
    in the target plasmid.
 +
</p>
 +
<p>
 +
    GFP sequences for Primer design and corresponding plasmids/colonies can be acquired from Jaqueline.
 +
</p>
 +
<p>
 +
    E1&amp;2:
 +
</p>
 +
<p>
 +
    Sequencing and restriction control were ok. Strains are frozen and documentation is being prepared.
 +
</p>
 +
<p>
 +
    Ask Silja for origin of the enzymes for better documentation. One is from Heiko Nacke, the other from Genis. In addition the enzyme sequences can be
 +
    Blasted for identification.
 +
</p>
 +
<p>
 +
    Phosphatase contains PstI, which is why currently it is not compatible with biobricks. In the future the restriction sites will have to be changed, if
 +
    submission should occur (PCR modification).
 +
</p>
 +
<p>
 +
    Dockerins:
 +
</p>
 +
<p>
 +
    Heat shock transformation did not work for any of the 4 dockerins, but positive control worked fine.
 +
</p>
 +
<p>
 +
    Vector was cut with EcoRI and KpnI as a test and KpnI was not very efficient. Due to this the restrictions were done separately.
 +
</p>
 +
<p>
 +
    Next step is a ligation and a gel to verify it immediately after to find reasons for failure:
 +
</p>
 +
<p>
 +
    - T4 ligase working?
 +
</p>
 +
<p>
 +
    - Phosphorylation issues -&gt; skip this step
 +
</p>
 +
<p>
 +
    - Find Plasmid formula to calculate ideal ligation ratio (depends on insert length and is usually 3:1) -&gt; overloading the vector may cause different
 +
    ligation variants
 +
</p>
 +
<p>
 +
    - Can E.coli express the dockerins?
 +
</p>
 +
<p>
 +
    Oder dockerins as codon optimised gBlocks from IDT, as a backup.
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu17').slideToggle(300, function callback() {  }); return false;"><h2>24.07.15</h2></a>
 +
 +
<div id="menu17">
 +
<p>
 +
    Present: Debbie, Avi, Julian, DEnnis
 +
</p>
 +
<p>
 +
    Excused: Verena, Steffi, Nida, Sindhu
 +
</p>
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Enzyme 3: Transformations didn't work, so the project will be restarted from PCR stage and a new ligation.
 +
</p>
 +
<p>
 +
    The sequencing is also not working, possibly due to wrong primers or wrong primer concentrations.
 +
</p>
 +
<p>
 +
    Dockerin/Scaffoldin: both scaffoldins are at the expression stage
 +
</p>
 +
<p>
 +
    Dockerins in gBlocks have arrived.
 +
</p>
 +
<p>
 +
    Old Dockerins may not be restrictable due to missing bands around the restriction sites.
 +
</p>
 +
<p>
 +
    New Plasmid isolation is on the way and work will continue on the dockerins as before.
 +
</p>
 +
<p>
 +
    <strong>2.) 3D Model:</strong>
 +
</p>
 +
<p>
 +
    Debbie has checked the sequences and has the Amino Acid sequences for all enzymes, but cannot find the scaffoldin sequences. When used they result in short
 +
    fragments. Dockerins do not work either due to too many stopcodons.
 +
</p>
 +
<p>
 +
    Double check the files and give her new files.
 +
</p>
 +
<p>
 +
    <strong>3.) Human Practices:</strong>
 +
</p>
 +
<p>
 +
    Julian will contact people at the Xlab.
 +
</p>
 +
<p>
 +
    A stop motion movie is being made that simply explains our project
 +
</p>
 +
<p>
 +
    A talk needs to be organised around our project and synthetic biology -&gt; Beer and Pretzels? Julian will contact some people and organise it.
 +
</p>
 +
<p>
 +
    Once returned from iGEM, the presenters will also present their project to the faculty at a faculty talk on the 27.10.2015
 +
</p>
 +
<p>
 +
    <strong>4.) Meetup:</strong>
 +
</p>
 +
<p>
 +
    The group that will travel to Marburg for the German Meetup will prepare short presentation
 +
</p>
 +
<p>
 +
    <strong>5.) Poster:</strong>
 +
</p>
 +
<p>
 +
    Dennis is happy to help with the organisation of the poster
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu18').slideToggle(300, function callback() {  }); return false;"><h2>18.08.15</h2></a>
 +
 +
<div id="menu18">
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Ligation and transformation of enzymes and dockerins with extra bases worked all around there are positive restriction results (but for 2). Plasmids have
 +
    been sent for sequencing.
 +
</p>
 +
<p>
 +
    - enzymes are being induced in fat medium for next day protein purification. Induction must be done with L-arabinose due to the pBAD vectore.
 +
</p>
 +
<p>
 +
    Scaffoldin: ACEL purification yielded 1mg/ml of Protein, but it still needs to be verified on an SDS gel.
 +
</p>
 +
<p>
 +
    Cellulase_CCEL construct is being sequenced.
 +
</p>
 +
<p>
 +
    <strong>2.) BioBricks:</strong>
 +
</p>
 +
<p>
 +
    Avril will design Primers with the proper restriction sites for the BioBrick shipping vector. EcoRI and PstI will be used.
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu20').slideToggle(300, function callback() {  }); return false;"><h2>24.08.15</h2></a>
 +
 +
<div id="menu20">
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Each section of labwork needs to be reassigned in order to optimize our work
 +
</p>
 +
<p>
 +
    Dennis &amp; Julian will work on Gelfiltrations and Protein extractions. They may need some help with all the inductions.
 +
</p>
 +
<p>
 +
    Alaa will make sure all protocols are written for the Wiki in time for the Wiki Freeze. Every group needs to write up their protocols and results.
 +
</p>
 +
<p>
 +
    Avril will make Primers for BioBricks of Dockerins with and without Enzymes and ask the Aachen Team for some Enzymes (design Primers for these as well)
 +
</p>
 +
<p>
 +
    Stefi will work on Poster and Presentation
 +
</p>
 +
<p>
 +
    Enzyme Dockerin constructs and BioBricks need to be continued and brought to work. See if we can find an enzyme cascade that we could attach to the
 +
    Flexosome.
 +
</p>
 +
<p>
 +
    <strong>2.) Finances:</strong>
 +
</p>
 +
<p>
 +
    An overview over our current finances has to be made to know what we have for Tshirts and labcosts.
 +
</p>
 +
<p>
 +
    <strong>3.) Safety Form:</strong>
 +
</p>
 +
<p>
 +
    The safety form deadline is this week. Avril will work on it and double check it with Heiko.
 +
</p>
 +
</div>
 +
<a href="" onClick=" $('#menu21').slideToggle(300, function callback() {  }); return false;"><h2>27.08.15</h2></a>
 +
 +
<div id="menu21">
 +
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Enzymes: Phosphatase activity test in different Buffer is working
 +
</p>
 +
<p>
 +
    Cellulase: Primers have not come yet
 +
</p>
 +
<p>
 +
    Induction: Pho_CTHE and rfp_ACEL are in fat medium and the spare amount will be used with the FPLC purification.
 +
</p>
 +
<p>
 +
    Primers have been designed for most constructs
 +
</p>
 +
<p>
 +
    Verena, Udhaya and Sindhu will continue the work on Dockerins, Est_BCEl and cjblue for BioBrick creation.
 +
</p>
 +
<p>
 +
    <strong>2.) Wiki:</strong>
 +
</p>
 +
<p>
 +
    Alaa and Udhaya have been working on the Method Collection for the Wiki
 +
</p>
 +
<p>
 +
    Ask Alex to make it pretty and insert the Aix-Marseille Gold Medal for collaboration
 +
</p>
 +
<p>
 +
    <strong>3.) ETC:</strong>
 +
</p>
 +
<p>
 +
    Steffi is working on Poster and Presentation. She has designed Tshirts with Verena and both have looked for enzyme pathways within the biobricks.
 +
</p>
 +
 +
</div>
 +
<a href="" onClick=" $('#menu22').slideToggle(300, function callback() {  }); return false;"><h2>03.09.15</h2></a>
 +
 +
<div id="menu22">
 +
<p>
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Protein purification of CTHE has been successful. Other constructs are not working. Not sure whether due to pBAD vector, therefore sequences need to be
 +
    checked.
 +
</p>
 +
<p>
 +
    Aachen Enzymes have arrived and the first BioBricks made (still need to be sequenced). BioBricks with colours amilCP, cjBlue and efforRed are being used as
 +
    backup.
 +
</p>
 +
<p>
 +
    Purification: Could the enzymes be deactivated by the purification?
 +
</p>
 +
<p>
 +
    Can we check scaffoldin interactions, even though enzymes may not function? So far it hasnt been possible to purify any of the enzymes or RFP and not to
 +
    show a 100% positive activity test.
 +
</p>
 +
<p>
 +
    - Restart expression tests
 +
</p>
 +
<p>
 +
    - start cellulase induction
 +
</p>
 +
<p>
 +
    - check RFP again with a control
 +
</p>
 +
<p>
 +
    - could it be possible to use the scaffoldin to purify the enzyme-dockerins?
 +
</p>
 +
<p>
 +
    - check for pBAD promoter error and ask Robert for other expression vectors.
 +
</p>
 +
<p>
 +
    - try and get some sort of interaction even if it is crude
 +
</p>
 +
<p>
 +
    - is there a BioBrick expression vector?
 +
</p>
 +
<p>
 +
    <strong>2.) Presentation:</strong>
 +
</p>
 +
<p>
 +
    First presentation meeting today
 +
</p>
 +
</div>
 +
<a href="" onClick=" $('#menu25').slideToggle(300, function callback() {  }); return false;"><h2>10.09.15</h2></a>
 +
 +
<div id="menu25">
 +
<p>
 +
<p>
 +
    <strong>1.) BioBricks:</strong>
 +
</p>
 +
<p>
 +
    Est-BCEL plasmid needs to be re-inoculated to get more plasmid. ACEL, BCEL and CCEL plasmids in the BioBrick vector look good and just need sequencing.
 +
</p>
 +
<p>
 +
    <strong>2.) Aachen:</strong>
 +
</p>
 +
<p>
 +
    HPS and PHI have been succesfully inserted into pBAD with sequencing being fine. Primers for expression in pET100 have arrived.
 +
</p>
 +
<p>
 +
    <strong>3.) Colours:</strong>
 +
</p>
 +
<p>
 +
    amilCP and eforRed have been sequenced but only amilCP is good, cjBlue is still in the BB vectore´
 +
</p>
 +
<p>
 +
    <strong>4.) Cellulase:</strong>
 +
</p>
 +
<p>
 +
    Restriction control in pBAD is good. Insertion into pET100 can be tried.
 +
</p>
 +
<p>
 +
    <strong>5.) pET100:</strong>
 +
</p>
 +
<p>
 +
    Esterase and Phosphatase have been inserted into pET100 with the dockerins. Need to wait if ligation and transformation has worked or if it needs to be
 +
    redone.
 +
</p>
 +
</p>
 +
</div>
 +
 +
 +
</html>
 +
 +
[[File:TeamGoettingen_notebook.jpg|center|250px]]
 +
<html>
 +
</div></div> <!--These are the closing tags for div id="mainContainer" and div id="contentContainer". The corresponding opening tags appear in the template that is {{included}} at the top of this page.-->
 
</html>
 
</html>

Latest revision as of 11:19, 18 September 2015



Notebook

15.12.14

08.01.15

13.01.15

15.01.15

19.01.15

02.02.15

16.02.15

18.02.15

23.02.15

16.03.15

23.03.15

07.04.15

13.04.15

21.05.15

19.06.15

25.06.15

24.07.15

18.08.15

24.08.15

27.08.15

03.09.15

10.09.15

TeamGoettingen notebook.jpg