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September 17th 2008

Miniprep

We did a miniprep on the culture of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115034 K115034], possibly we will try to have it sequenced, although it's a bit long. The end concentration was ca. 300 ng/ul in 100 ul.

Transformation

Due to failing of the previous transformations of our end RNA thermometer testing parts, we've started new ligations, which we transformed today.

Gel analysis of previous miniprep

Due to the failing of the transformation yesterday, we wanted to put the cut and purified DNA on gel. This can be seen in figure 1.

Figure 1. Restriction of the previous 3A-assemblies. A marker has been loaded twice.

It can be seen the restrictions of 12 September are far from complete, which also indicates why the ligations/transformations didn't work. The restrictions of 16 September are better, but next time we will cut quantities of over 1000 ng (pSB1AT3 & R0080) vector with 1 ul per restriction enzyme. Furthermore K115032 and K115033 look incompletely cut, so we won't expect very good transformations of these parts. These two parts are however highly similar to K115029 and K115030, so these two constructs are the least important ones to measure.

Growing for luciferase assay

As we have the control and a thermosensitive part, we'll start growing cultures today to do an assay on Friday. We want to test what will be the fastest way for obtaining samples, therefore we started a range of cultures, both growing conditions are on K115034 (designed to switch @ 27ºC) and K115012 (reference). All cultures grown were shaken with a 1 inch stroke at 175 rpm.

• Growing overnight with arabinose induction at room temperature
• Growing overnight with arabinose induction at 37ºC
• Growing overnight without induction at 37ºC (to produce biomass), induce next day for 5 hours at room temperature (to produce luciferase)
• Growing 2 times overnight (ca. 36 h) with arabinose induction at room temperature
• There was one extra sample of K115012 to test R0080 for leakiness. This sample was grown o/n at 37ºC without arabinose induction.

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