Design
Build
Test |
Once you have designed and built your new genetic parts, you need to test them for function. In this case, you would combine each tandem promoters with an RBS, a reporter gene, and a terminator to form a testing transcriptional unit (TU). Note: Before beginning the flow setup, you should make sure you have chosen a fluorescent gene that you know how to test (refer to the Interlab Study page). After creating the TUs, you will follow a flow cytometry workflow to test the induction or repression of each promoter and also the promoters in tandem. The flow setup takes place over three days. Attached is a completed protocol for a flow cytometry tandem promoter experiment. On the first day, you will streak out the controls and your constructs onto plates with the correct antibiotic resistance. For controls, you will use J23104RM (positive RFP control), J23104GM (positive GFP control), COXGR (co-expression GFP RFP), COXRG (co-expression RFP GFP), and a strain control (Bioline or Pro strain). When testing inducible tandem promoters, it is advantageous to use the Pro strain because it contains araC, lacI, and tetR. This prevents you from having to build additional transcriptional units to express these genes. In the Pro strain, the tandem promoters will, by default, be in their off state.
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 Figure 1: Plate Setup |
| After streaking out all the necessary parts, you should fill in the Plate Setup tab on the protocol Excel sheet. You will be testing each construct in triplicate. For your constructs, there should be at least four columns per small molecule for each TU and four columns for the combination of small molecules for each construct. The first three columns are for the three different colonies (each row is a different concentration), and one column is for the negative control (media, but no construct). It is recommended that you leave one blank column in between your last colony and the negative control column to prevent your negative from being contaminated. There also needs to be a controls plate with four cells per control. Figure 1 shows an example of plate setup. After growing your plates overnight, you will pick three colonies from each plate for eight hours in LB + antibiotic media. These cells will be grown in deep well plates inside a shaker. While these are growing, you should fill in the Media Setup tab on the protocol. You will have to decide on the range of concentrations you want to use for each small molecule. If your constructs have been tested before, look through the literature for ideas on which concentrations to test. The Media Setup page calculates how much LB media and stock solution to add in order to get each concentration of small molecule. After eight hours, you should check the deep well plates for growth before beginning the next step. If the controls and constructs have growth, set up the small molecule plates by following the instructions on the Plate Setup tab. |
 Figure 2: TASBE Tools |
| The following day is testing day! Before running your samples through the flow, dilute your overnight plates with PBS (instructions are on protocol excel sheet). You will need to run all of your setup plates through the flow following standard operations procedure for high throughput testing in the Flortessa. More information for this procedure can be found on our Interlab Study page. After completing your experiment, you should analyze the data using the TASBE tools (refer to Software Tools page) as shown in Figure 2. From the TASBE tools, you should get graphs that look similar to Figure 3 and Figure 4. You should expect to see increasing fluorescent expression as the small molecule concentrations increase. Ideally, there should be a wide range of expression for each small molecule and for the two small molecules together. If your graph has small or no range of fluorescence, you should redo the flow experiment with different concentrations of small molecules. You can also redo the experiment to tighten error bars. Additionally, you can try testing your genetic part with a variety of different genetic part variations. For example, you could change the RBS used in your transcriptional unit to see the effect on your fluorescent expression. One quick way of doing this is multiplexing. This will help you decide which genetic parts to use in your final complex genetic device. |
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| Figure 1: Flow Cytometry graph for pTet-pBad level 1 construct with RFP for three conditions: atc (red), arabinose (blue), atc and arabinose (purple) |
Next Steps
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