Team:Warsaw/Calendar-Stage1/29 August 2010

From 2010.igem.org

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<br> 30.08.2010 Monday
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<br><br>Note:We improved the way of cell preparation by using lysozyme or triton to reduce the adherence of cells
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<br><br><div class="note"> Previous 2 days: Set up and culturing Hela cell culture [Kamil]</div>
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<br> Conducted according to the protocol included in "Quantitative measurement of mamallian cell invasion by bacteria using flow cytometry" standard (<a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_59:_Quantitative_measurement_of_mamallian_cell_invasion_by_bacteria_using_flow_cytometry">RFC 59 standard</a> point 5)
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<br>
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<br><div class="note"> Previous day: Set up liquid cultures of E.coli with different constructs [Ania P.]</div>
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<br> Conducted according to the protocol included in "Quantitative measurement of mamallian cell invasion by bacteria using flow cytometry" standard (<a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_59:_Quantitative_measurement_of_mamallian_cell_invasion_by_bacteria_using_flow_cytometry">RFC 59 standard</a> point 5)
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<br>
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<br><div class="note"> Testing the invasivnes of E.coli in Hela cells [Kamil,Cherry]</div>
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<br>1. Incubation of Hela cells with different E.coli with different constructs.
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<br>2. Preparation of samples with incubated cells and examining them in flow cytometer.
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<br>Conducted according to the protocol included in "Quantitative measurement of mamallian cell invasion by bacteria using flow cytometry" standard, but with an extra version where we used Triton instead of lysozyme(<a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_59:_Quantitative_measurement_of_mamallian_cell_invasion_by_bacteria_using_flow_cytometry">RFC 59 standard</a> points 6 and 7)<br>
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<br><div class="note"> Data analysis with Cell Quest and Microsoft Excel [Cherry]</div>
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<br>
<br /><strong>legend:</strong>
<br /><strong>legend:</strong>
<hr />
<hr />
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<br /><strong>E.coli constructs:</strong>
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<br /><strong>E.coli TOP 10 constructs:</strong>
<br />G- GFP
<br />G- GFP
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<br />J- JPH+Listeriolysin O+Invasin+GFP
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<br />J- J23102+Listeriolysin O+Invasin+GFP
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<br />A- APH+Listeriolysin O+Invasin+GFP
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<br />A- pAraC+Listeriolysin O+Invasin+GFP
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<br />TOP- without construct
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<br />TOP- TOP10 without construct
<br />
<br />
<br />LIZ- treated with lysozyme
<br />LIZ- treated with lysozyme
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<br />
<br />
<br /><strong>bacteria:</strong>
<br /><strong>bacteria:</strong>
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<br />30-08-2010.001- TOP
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<br />30-08-2010.001- TOP10 without construct
<br />30-08-2010.003- G
<br />30-08-2010.003- G
<br />30-08-2010.004- J
<br />30-08-2010.004- J
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<br />30-08-2010.043- TOP LIZ
<br />30-08-2010.043- TOP LIZ
<br />30-08-2010.044- G LIZ
<br />30-08-2010.044- G LIZ
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<br />
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<br /><br />cytometer plots:
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<br />
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<br>FSC- Forward Scatter, cell size
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<br>SSC- Side Scatter, cell complexity
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<br>FL-1- FL-1 sensor, cell fluorescence
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<br><br />
<hr />
<hr />
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<br />
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<br /><strong>Cytometer data from Cell Quest analysis:</strong>
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<img src="https://static.igem.org/mediawiki/2010/9/90/Bacto.30.08_01.png" width="500px" />
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<br><img src="https://static.igem.org/mediawiki/2010/9/90/Bacto.30.08_01.png" width="500px" />
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<img src="https://static.igem.org/mediawiki/2010/8/81/Bacto.30.08_02.png" width="500px" />
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<br>
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<br>Lysozyme treatment:
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<br><img src="https://static.igem.org/mediawiki/2010/8/81/Bacto.30.08_02.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/e/ec/Bacto.30.08_03.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/e/ec/Bacto.30.08_03.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/b/bf/Bacto.30.08_04.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/b/bf/Bacto.30.08_04.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/e/e1/Bacto.30.08_05.png" width="300px" />
<img src="https://static.igem.org/mediawiki/2010/e/e1/Bacto.30.08_05.png" width="300px" />
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<br>
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<br>Triton treatment:
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<br>
<img src="https://static.igem.org/mediawiki/2010/5/5f/Bacto.30.08_06.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/5/5f/Bacto.30.08_06.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/8/8e/Bacto.30.08_07.png" width="500px" />
<img src="https://static.igem.org/mediawiki/2010/8/8e/Bacto.30.08_07.png" width="500px" />
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<br />
<br />
<br />
<br />
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<hr /><br />
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<strong>Data from Cell Quest processed with Microsoft Excel:</strong>
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<br /><br /><br />
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<br>TOP- Hela cels with regular TOP E.coli
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<br>M1,M2,M3,M4- PIN Units (according to <a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_59:_Quantitative_measurement_of_mamallian_cell_invasion_by_bacteria_using_flow_cytometry">RFC 59 standard</a> point 7)
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<br><br><img src="https://static.igem.org/mediawiki/2010/4/4e/Bacto.30.08_08_table.png" />
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<br />
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<br />
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Latest revision as of 20:23, 27 October 2010

Example Tabs


30.08.2010 Monday

Note:We improved the way of cell preparation by using lysozyme or triton to reduce the adherence of cells

Previous 2 days: Set up and culturing Hela cell culture [Kamil]

Conducted according to the protocol included in "Quantitative measurement of mamallian cell invasion by bacteria using flow cytometry" standard (RFC 59 standard point 5)

Previous day: Set up liquid cultures of E.coli with different constructs [Ania P.]

Conducted according to the protocol included in "Quantitative measurement of mamallian cell invasion by bacteria using flow cytometry" standard (RFC 59 standard point 5)

Testing the invasivnes of E.coli in Hela cells [Kamil,Cherry]

1. Incubation of Hela cells with different E.coli with different constructs.
2. Preparation of samples with incubated cells and examining them in flow cytometer.
Conducted according to the protocol included in "Quantitative measurement of mamallian cell invasion by bacteria using flow cytometry" standard, but with an extra version where we used Triton instead of lysozyme(RFC 59 standard points 6 and 7)

Data analysis with Cell Quest and Microsoft Excel [Cherry]


legend:

E.coli TOP 10 constructs:
G- GFP
J- J23102+Listeriolysin O+Invasin+GFP
A- pAraC+Listeriolysin O+Invasin+GFP
TOP- TOP10 without construct

LIZ- treated with lysozyme
TRIT- treated with triton
X- untreated

bacteria:
30-08-2010.001- TOP10 without construct
30-08-2010.003- G
30-08-2010.004- J
30-08-2010.005- A

Hela cells with bacteria:
30-08-2010.033- X TRIT
30-08-2010.034- A TRIT
30-08-2010.035- J TRIT
30-08-2010.036- A LIZ
30-08-2010.037- J LIZ
30-08-2010.038- without bacteria TRIT
30-08-2010.039- TOP TRIT
30-08-2010.040- G TRIT
30-08-2010.041- X LIZ
30-08-2010.042- without bacteria LIZ
30-08-2010.043- TOP LIZ
30-08-2010.044- G LIZ

cytometer plots:
FSC- Forward Scatter, cell size
SSC- Side Scatter, cell complexity
FL-1- FL-1 sensor, cell fluorescence



Cytometer data from Cell Quest analysis:


Lysozyme treatment:


Triton treatment:





Data from Cell Quest processed with Microsoft Excel:



TOP- Hela cels with regular TOP E.coli
M1,M2,M3,M4- PIN Units (according to RFC 59 standard point 7)