Team:BostonU/ChimeraExample

From 2014.igem.org

• TEAM
• PROJECT
  • Overview
  • Priority Encoder
  • MoClo Assembly Method
  • Low Copy Backbones
  • Tandem Promoters
  • Fusion Proteins
  • Repressor Proteins
  • Multiplexing
  • BioDesign Automation Tools
  • Future Work
• ACHIEVEMENTS
  • Data Collected
  • Parts Submitted
  • Chimera Workflow
  • Chimera Example
  • Medal Fulfillment
• NOTEBOOK
  • Training
  • Protocols
  • Backbones
  • Tandem Promoters and Repressors
  • Fusion Proteins
• CONSIDERATIONS
  • Collaborations
  • Measurement Track
  • Safety
  • Interlab Study
  • NEGEM
  • Policy and Practices
• ACKNOWLEDGEMENTS

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. 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). 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. A more streamlined version of Eugene is also available, called miniEugene, which can also be used for this step. Below, we show files for both Eugene and miniEugene with graphic outputs (made automatically via Pigeon) and statistics for miniEugene, which is more user friendly for wet lab researchers unfamiliar with coding languages. For the tandem promoters alone as parts, here are the design files. Eugene: Tandem Promoters Only miniEugene: Tandem Promoters Only Below is the output SBOL Visual graphic for the miniEugene file above, along with the miniEugene statistics for the file. Figure 3: The 6 possible outcomes of the tandem promoters with pBad, pLac, and pTet (right) and the miniEugene statistics, including final device output. For the tandem promoter Test Devices, here are the design files. Eugene: Test Device for Tandem Promoters miniEugene: Test Device for Tandem Promoters Below is the output SBOL Visual graphic for the miniEugene file above, along with the miniEugene statistics for the file. Figure 4: The 6 possible outcomes of the tandem promoter Test Device with RFP (right) and the miniEugene statistics, including final device output.

Build Step 1: Now with the designs flushed out with Eugene or miniEugene, you can begin the assembly of the tandem promoters. Raven can be used to design oligos and generate an assembly plan, based on your assembly method of choice. You need to input the sequence data for the basic parts into Raven and select the assembly method. For this example, MoClo was chosen as the assembly strategy. 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. Here is the Raven input file for generating the tandem promoters (Figure 4): Tandem Promoter Raven Here is the Raven input file for generating the tandem promoter Test Devices (Figure 5): Tandem Promoter Test Devices Raven Figure 5: Raven assembly graph output for the six tandem promoters combinations between pBad, pLac, and pTet. This graph was generated with the MoClo assembly method selected in the Raven software. Figure 6: Raven assembly graph output for the six tandem promoters Test Devices containing RFP as the output fluorescent protein. This graph was generated with the MoClo assembly method selected in the Raven software. Step 2: Following the assembly plan generated by Raven, the tandem promoters are cloned. This may involve PCR steps, restriction digests, and/or ligations depending on the assembly method the user selected. For the MoClo assembly, this involves running PCR reactions using oligos that can be designed by Raven and then cloning the new parts into destination vectors through a one-pot digestion-ligation reaction. Once the tandem promoters are complete, the Test Devices must also be built following the Raven assembly graph. Step 3: Any new constructs, including the tandem promoters and subsequent Test Devices, should be sequence verified prior to moving on to the Test stage.

Test Step 1: Given the design selection of pBad, pTet, and pLac as potential input promoters for the tandem promoters, the published small molecule induction curves need to be researched prior to running flow cytometry. Since the literature results will often show different concentrations of the small molecules (arabinose for pBad, anhydrotetracycline for pTet, and isopropyl β-D-1-thiogalactopyranoside for pLac), you should expect to run a wide range of small molecules to determine which range will work best for your particular parts. Step 2: 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). 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 7: 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 2 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 8: TASBE Tools home page (https://synbiotools.bbn.com/), showing linking for entering data and running experiments on the left hand side. 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. Step 3: After completing your experiment, you should analyze the data using the TASBE tools (refer to Software Tools page) as shown in Figure 8. From the TASBE tools, you should get graphs that look similar to Figure 9 and Figure 10. 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 9: Flow Cytometry graph for pTet-pBad level 1 construct with RFP for three conditions: atc (red), arabinose (blue), atc and arabinose (purple)

Figure 10: Flow Cytometry graph for pBad-pTet level 1 construct with RFP for three conditions: atc (red), arabinose (blue), atc and arabinose (purple)

Next Steps

After testing the constructs and confirming their function, you can proceed to building similar parts based on the final device design that Eugene output initially. For the priority encoder, you will need to make tandem promoters for many different regulators. After confirming the sequences for all required parts, it is important to make sure that you put the right parts in the right positions. Eugene will only give you blank positions. The next phase is about filling those positions with parts that work the best. This can be done by a multiplexing reaction . One reaction will yield many possible constructs and you should then pick as many colonies as possible and test all of them using flow cytometry. This will allow you to pick the colony that has the best transfer curve. This is done with as many transcriptional units (TUs) as possible. We will use the following testing strategy for the Priority Encoder. After deciding which units work best independently, you should test several combinations of more than two TUs to test the efficacy of their regulatory arcs. Based on this data, generate a Eugene file for the final device to permute all possible outcomes of the chosen optimal TUs. Then, generate a Raven file and clone your final device based on the strategy Raven designs. Sequence confirm the final design and test using Flow Cytometry.

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