Team:UGent/Experiments
From 2013.igem.org
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Besides measuring fluorescence, we also measure OD as this an indicator for the growth of the bacteria. This has to be taken into account when analysing fluorescence results, since fluorescence is also dependent of bacterial density. To compare bacterial colonies, grown in different IPTG concentrations, this factor has to be eliminated. That is why for comparison we will use fluorescence/OD. | Besides measuring fluorescence, we also measure OD as this an indicator for the growth of the bacteria. This has to be taken into account when analysing fluorescence results, since fluorescence is also dependent of bacterial density. To compare bacterial colonies, grown in different IPTG concentrations, this factor has to be eliminated. That is why for comparison we will use fluorescence/OD. | ||
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- | We used two protocols for this experiment. In the first version bacteria are grown for approximately 9 hours inside the | + | We used two protocols for this experiment. In the first version bacteria are grown for approximately 9 hours inside the FLUOstar and fluorescence and OD are measured each five minutes. The second protocol is less time consuming and consists of growing our bacteria in microtiter plates during 4 hours at 37°C (not in FLUOstar) after which fluorescence and OD are measured one time each inside the FLUOstar. |
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<A HREF="https://static.igem.org/mediawiki/2013/7/7d/UGent_2013_Protocol_Experiment_5.1.pdf" target="_blank">Protocol experiment 5.1</A> | <A HREF="https://static.igem.org/mediawiki/2013/7/7d/UGent_2013_Protocol_Experiment_5.1.pdf" target="_blank">Protocol experiment 5.1</A> |
Revision as of 02:22, 4 October 2013
ExperimentsTo test our idea we are conducting 6 experiments. These are described below. Experiment 1
Protocol experiment 1 Experiment 2
Experiment 3
Experiment 4Strains constructed in experiment 3 will be used to perform CIChE. Tandem gene replication of reporter protein GFP will be induced by replicating the antitoxin CcdA as a response on titration of the toxin CcdB. Titration of CcdB under inducible T7-promoter will be accomplished by different levels of IPTG [0.01 mM – 0.5mM] and different plasmid copy numbers [p5, p10 and p20]. Protocol experiment 4Experiment 5Through experiment 4 we obtained strains that underwent different levels of chromosomal evolution and should contain different construct copy numbers. In experiment 5 we will assess the number of duplications by growing our colonies in microtiter plates and measuring fluorescence intensity and optical density (OD) inside the FLUOstar OPTIMA (BMG LABTECH). Bacteria with higher construct copy numbers also contain a higher number of gfp copies, resulting in higher fluorescence intensities.
Protocol experiment 5.2 Experiment 6In this experiment, a new part, called BBa_K1105000, will be constructed by cloning LacIq-T7ccdB with standard Biobrick prefix and suffix in pSB1C3. Laciq-T7ccdB is derived from the plasmid p10-LacIq-T7ccdB, which originally comes from a mini F-plasmid positive strains (such as E. coli F+). The function of this part is to produce CcdB (in control of a T7 promotor), which interferes with the topoisomerase unit gyrA. Protocol experiment 6
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