Team:EPF-Lausanne/Our Project/T7 promoter variants/lysis

From 2011.igem.org

(Difference between revisions)
(Experimental Setup)
(Introduction)
 
(8 intermediate revisions not shown)
Line 6: Line 6:
=== Introduction ===
=== Introduction ===
-
We cloned the Berkeley Lysis cassette ([http://partsregistry.org/Part:BBa_K112808 BBa_K112808]) under control of the T7 promoter into a low copy number plasmid ([http://partsregistry.org/Part:pSB3K1 pSB3K1])
+
We cloned the Berkeley Lysis cassette ([http://partsregistry.org/Part:BBa_K112808 BBa_K112808]) under control of the T7 promoter into a low copy number plasmid ([http://partsregistry.org/Part:pSB3K1 pSB3K1]).
 +
[[File:t7_rbs_lysis_term.png]]
We tested lysis of cells haboring this plasmid in a platereader experiment. Lysis resulted in a drop in optical density after induction with IPTG. We observed a strong dependence on IPTG concentration.
We tested lysis of cells haboring this plasmid in a platereader experiment. Lysis resulted in a drop in optical density after induction with IPTG. We observed a strong dependence on IPTG concentration.
 +
 +
To learn more about how IPTG induction tests work, click [[EPF-Lausanne/Our Project/T7 promoter variants/lysis/iptg|here]].
=== Experimental Setup ===
=== Experimental Setup ===
Line 26: Line 29:
=== Results ===
=== Results ===
-
[[Image:lysis_dynamics.png|500px|center]]
+
[[Image:lysis_dynamics.png|600px|center]]
-
Data was produced in triplicate via the 96 well plate. This graphic shows the mean over the three data points for the measurements taken every 10 minutes. The values on the y-axis are relative fluorescence units (RFU) normalized by optical density (OD), while the x-axis represents time. The curves indicate that maximal lysis is produced with 500 uM IPTG, and that lesser degrees of lysis can be obtained with smaller doses of IPTG.   
+
Data was produced in triplicate via the 96 well plate. This graphic shows the mean over the three data points for the measurements taken every 10 minutes, with the exception of the 250 uM curve for which only two data points were available for the time evolution. The values on the y-axis are optical density (OD) at 600nm, while the x-axis represents time. The curves indicate that maximal lysis is produced with 500 uM IPTG, and that lesser degrees of lysis can be obtained with smaller doses of IPTG.   
-
[[Image:dose_response.png|500px|center]]
+
[[Image:dose_response.png|600px|center]]
-
The dose-response graph above was also made using triplicate data. This time, the functional T7-lysis plasmid (C2) was tested against two negative controls: a T7-RFP plasmid (which should only fluoresce, not lyse) and a plasmid containing a non-functional lysis cassette driven by a T7 promoter (C11). The points of each curve are the unaveraged fluorescence responses (normalized by OD) over the last hour of the experiment, which we take to be a good estimate of the steady-state saturation. The C2 lysis plasmid shows strong lysing, since the fluorescence drops significantly as the IPTG dose increases.  
+
The dose-response graph above was also made using triplicate data. This time, the functional T7-lysis plasmid (C2) was tested against two negative controls: a T7-RFP plasmid (which should only fluoresce, not lyse) and a plasmid containing a non-functional lysis cassette driven by a T7 promoter (C11). The points of each curve are the OD values of each replicate averaged over the last hour of the experiment. For the lysis plasmid we observed a strong lysis response with increasing IPTG concentrations. Both controls show no significant change in endpoint OD in respect to IPTG concentration.
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 20:57, 21 September 2011