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September 19th 2008

Luciferase Assay

Today we've done our first luciferase assay. The samples we had in the end were either K115034 (34) or K115012 (12, control).

Samples

A: 12, Growing overnight with induction at room temperature, lysed by sonification
B: 34, Growing overnight with induction at room temperature, lysed by sonification
C: 12, Growing overnight with induction at 37oC, lysed by sonification
D: 34, Growing overnight with induction at 37oC, lysed by sonification
E: 12, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
F: 34, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
G: 12, Growing 2 times overnight with induction at room temperature, lysed by sonification
H: 34, Growing 2 times overnight with induction at room temperature, lysed by sonification
I: 12, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
J: 34, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
K: 12, Growing o/n at 37oC without induction, to test R0080 for leakiness, lysed by sonification

Protocol

The protocol used in the case of most samples, when lysed by sonification was:
1. Grow as indicated above and induce immediately, unless stated otherwise
2. Pellet 3 ml of cells
3. Resuspend in 100 ul 15 mM Tris HCl
4. Destroy by sonification (3 * 15 s)
5. Freeze at -20
6. Add 20 ul of lysed cell sample to a well
7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)

If lysis by kit buffer (Promega) was used, the protocol became:
1. Grow as indicated above and induce immediately, unless stated otherwise
2. Pellet 3 ml of cells
3. Resuspend in 100 ul 1X lysis buffer
4. Leave at room temperature for 15-30 minutes
5. Freeze at -20
6. Add 20 ul of lysed cell sample to a well
7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)

Results

After a lot of fine tuning of the luminometer, the luminescence values were obtained as shown in graph 1.

Graph 1. Luminescence corrected for cell density (OD=1) at the time of sample preparation.

The OD of the various samples were all around 0.7, with the exception of A and B, which were only grown o/n at room temperature. Their OD was ca. 0.15.

Although not very consistent, these results indicate that the thermosensitve K115034 part has lower expression of luciferase, looking at sample I and J. However, a similar ratio between K115034 and K115012 is found for 37oC (sample C and D). These samples at 37oC were only lysed by sonification, and this shows to give very inconsistent results (looking at sample E and F, which were identical in treatment and OD at sampling time).

In conclusion, there might be some effects, but we'll need to do new experiments lysing all samples with the kit sample buffer. Also we should grow samples on the same marker, as we've done this experiment on both T and K resistance. Since both vectors also contain an ampicillin resistance, we should grow on ampicillin. Finally we still need to measure total protein content, as this is a better reference for protein content as OD, which we've used now for correction.

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