Team:BostonU/ChimeraExample

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Chimera working example
As described here , the tandem promoters were an integral part in designing the Chimera Workflow for building large devices and specifically, the priority encoder . In this page, we describe the detailed methodology that you could use to design, build and test tandem promoters. The same could be applied to any basic or complex device. Below, where we indicate "Step 1", those steps refer to steps outlined in the Chimera Workflow.

Design


    Step 1: Based on the breakdown of the priority encoder (the final device desired), tandem promoters that can be controlled with small molecules are easily identified as new basic parts that are required to make the priority encoder. Three controllable promoters (pBAD, pTetR, and pLacI) are well studied in the literature and will be included in the Eugene file (Design, Step 3).
    Figure 1: Basic design for tandem promoters

    Step 2: In order to measure fluorescence with tandem repressible promoters, you will need a Test Device where the tandem promoters control the expression of a reporter fluorescent protein. One major element that must be considered when designing this Test Device is the testing strain. For this example, the testing strain you will use in this example is an E. coli DH5-alpha Pro strain where araC, tetR, and lacI are constitutively expressed in the genome. This impacts the Test Device design heavily, since the design becomes much more simplified since the Test Device does not need to include the expression of these repressor proteins.
    Figure 2: Testing contruct built to test for function of tandem promoters

    Step 3: Once the basic parts are identified, you need to input the tandem promoter parts and Test Device that you want in your design into a Eugene file. Eugene will output all possible permutations of those parts. To obtain the final design of the desired constructs, add constraints to Eugene. Adding a few basic constraints greatly reduces the number of possible designs. This will help you decide which promoters you want to assemble in tandem and in what order.

    Here is the final Eugene file for this design: Test Device for Tandem Promoters

Build


Thereon, you can begin the assembly of the promoters. Input the sequence data for the promoters that you decided to use into Raven. Raven designs oligos that can then help you use Polymerase Chain Reaction (PCR) to create flanking regions based on your assembly method. You can then ligate the promoters together.
If you use MoClo to do this, the oligos designed by Raven will help you create a new fusion site. The placement of the fusion site will depend on the order of the two promoter sequences. After assembling the tandem promoters, it is integral that you test whether the function of one promoter impacts that of the other. For this, you need to build transcriptional units with the tandem promoter and a reporter protein (Figure 2). Then all necessary inputs are made into Raven, it will output a fast, cost-effective building strategy using you desired assembly method. This construct can then be tested using flow cytometry as described below. Any new constructs should be sequence verified.








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.

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.
Figure 3: Flow Cytometry graph for pTet-pBad level 1 construct with RFP for three conditions: atc (red), arabinose (blue), atc and arabinose (purple)
Figure 4: Flow Cytometry graph for pBad-pTet level 1 construct with RFP for three conditions: atc (red), arabinose (blue), atc and arabinose (purple)

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