"
Team:Warsaw/Calendar-Stage1/29 August 2010
From 2010.igem.org
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)
