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September 25th 2008

Luciferase Assay

Sample preparation

All the cultures growing had reached an OD of about 0.4 in the morning. When 3 hours later the OD's had stayed practically constant, we lysed them. Also the cultures of K115036 growing at 30oC and 37oC have been lysed. Also has K115036 been pelleted and frozen at -20 for miniprepping later.

Of all cultures lysed, 10x and 100x dilutions have been made in H2O. 100x dilutions showed to be in the linear range of the BCA protein assay in a preliminary test, so these were used for total protein determinations.

Results

The strains measured contained one of the following constructs: P1010 in pSB1AT3, K115012, K115029, K115031, K115032 (which had a dubious PCR product, it was lane 32 1' here), K115034 and K115036. Only K115036 was not measured at 26oC.

Unfortunately the reader had some problems, making it impossible to do good measurements on K115031. Also did we find out pure 100x diluted lysis buffer gives quite high values in the total protein assay, so in next experiments we'll need to correct our calibration curve for this as well. In an extra test of R0080, we grew some non-induced samples at 37oC (K115034 and K115012), but these also had high expression of luciferase, so it will not be possible to grow and induce at different temperatures with R0080. The normalized results of the luciferase assay (corrected for protein content) are displayed in graph 1.

• N.B.: Before October 13, we normalized all the datapoints for luminescence of strain K115012 at the same temperature. After some discussion we decided it would be better to normalize luminescence per strain, for lowest available temperature.

Graph 1: Normalized Luciferase Activity measurements.

It can be seen that relative expression goes down from 37oC to 30oC. This is what we would hope to see. Strangely, expression goes up again at lower T. This might be a result of different growing conditions, as 37oC and 30oC were grown in a stove, whereas 26oC and 42oC were grown in a water bath. At high temperature, K115012 seems to remain a lot more stable than the thermosensitive constructs. More measurements must be done to obtain more datapoints at different temperatures to verify the observed behaviour between 37oC and 30oC. All normalized activities at 26oC are still lower than those at 37oC so that also indicates a temperature effect.

Miniprep K115036

To obtain a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115036 K115036] plasmid stock, we've frozen a cell pellet for later miniprepping.

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