Difference between revisions of "Team:HKUST-Rice/Expression"
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<br><br>For every single characterization, same procedures were followed. First, the sample which would be measured had to be inoculated in Falcon tubes. In the next day, 25-fold dilution was carried out for the inoculated samples by using the M9 minimal medium with specific inducers and concentrations. They would then be transferred into 96-well deep well plate for overnight induction. Again, the sample would be further diluted by ten-fold dilution in the next day. Several hours were needed to let the cells grow from lag phase to log phase. The OD600 should be around 0.4-0.7 ideally and kept constant for every trial. Ultimately, the result could be gathered with the help of EnVision® Multilabel Reader(OD595) using filter 485/14nm FITC and 535/25nm FITC for excitation and emission measurement respectively. All data would be plotted as graphs for further analysis. | <br><br>For every single characterization, same procedures were followed. First, the sample which would be measured had to be inoculated in Falcon tubes. In the next day, 25-fold dilution was carried out for the inoculated samples by using the M9 minimal medium with specific inducers and concentrations. They would then be transferred into 96-well deep well plate for overnight induction. Again, the sample would be further diluted by ten-fold dilution in the next day. Several hours were needed to let the cells grow from lag phase to log phase. The OD600 should be around 0.4-0.7 ideally and kept constant for every trial. Ultimately, the result could be gathered with the help of EnVision® Multilabel Reader(OD595) using filter 485/14nm FITC and 535/25nm FITC for excitation and emission measurement respectively. All data would be plotted as graphs for further analysis. | ||
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| − | < | + | <p style="font-size:200%"><u> Preparing medium concentration </u></p> |
| − | + | <p>M9 minimal medium was used for inoculation, making medium with inducers and dilution. It was chosen because of its low auto-fluorescence. Serial dilution was usually adopted for making medium with different concentration of inducers. | |
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Revision as of 12:29, 29 August 2015
Signal Co-expression
Expression platform
To examine the possible effects of co-expression, comparison between dose dependent fluorescence response expressed by one construct in a single plasmid and two constructs in one plasmid was conducted . The purpose of the comparison relates to the design of nitrate, potassium ion and phosphate ion (NPK) sensor, which aims to combine three constructs characterised by different outputs within one plasmid.
As the substitute of the NPK inducible promoters, well-characterised promoters araBADp and lacZp were used as their biological mechanisms are better understood.
The construction of a double construct araBADp-lacZp allowed for a comparison between GFPmut3b and RFP measurements while changing concentrations of inducers. Two graphs of fluorescence changes according to different inducers concentrations for both single and double constructs could be analysed and compared.
Method
Cloning
There were two inducible single construct for characterization. They were araBADp with GFPmut3b and lacZp with mRFP induced by arabinose and IPTG respectively. Both of the constructs were built by digestion and ligation method following the RFC10 standard.
Same approach was also adopted for the double construct to ligate araBADp and lacZp together.
Experiment that we did
Characterization
As all of them were inducible constructs, fluorescence would only be expressed when inducers were present.
For every single characterization, same procedures were followed. First, the sample which would be measured had to be inoculated in Falcon tubes. In the next day, 25-fold dilution was carried out for the inoculated samples by using the M9 minimal medium with specific inducers and concentrations. They would then be transferred into 96-well deep well plate for overnight induction. Again, the sample would be further diluted by ten-fold dilution in the next day. Several hours were needed to let the cells grow from lag phase to log phase. The OD600 should be around 0.4-0.7 ideally and kept constant for every trial. Ultimately, the result could be gathered with the help of EnVision® Multilabel Reader(OD595) using filter 485/14nm FITC and 535/25nm FITC for excitation and emission measurement respectively. All data would be plotted as graphs for further analysis.
Preparing medium concentration
M9 minimal medium was used for inoculation, making medium with inducers and dilution. It was chosen because of its low auto-fluorescence. Serial dilution was usually adopted for making medium with different concentration of inducers.
Result obtained
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Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per. Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.