Team:TEC COSTA RICA/Wet Lab
Wet Lab
The wet-lab characterization of our genetic suicide circuit serves as a proof of concept for the proposal presented in the description.
Goals
We set three main goals for our wet-lab work:
- Characterizing the behavior of our counter both associated to pBAD and pnrd.
- Characterizing the behavior of our overcount prevention system on itself and associated with the counter circuit.
- Obtaining our own recombination rate for the two selected recombinases for the optimization of the math model.
Constructs
Hence, four separate constructs were designed:
- Counter circuit
- Overcount prevention circuit
- Recombinase 1 characterization circuit
- Recombinase 2 characterization circuit
You can check out the parts we used in Parts.
Description
Our math modelling was used as reference for the specific RBS-sequence pairings, specifically regarding the RDF - recombinase ratio as well as the recombinase order. The biobrick system was chosen because of our need to use parts from the distribution making the assembly incompatible with either golden gate and gibson methods. A restriction site for HindIII was added to the counter and overcount constructs in order to make them compatible with each other and for different promoters to be tried out with the counter system. This also allows further modifications and optimizations.
Fragments
The fragments designed for this assembly are:
COUNT1: pBAD + site1A + site2A + BXB1
COUNT2: RFP + site1B + BXB1 RDF rev
COUNT3: TP901 + GFP + site2B + TP901 RDF rev
OVERC1: ACT + OPER SEC + RFP
OVERC2: IND PROM+ REPRESS
REC1: BXB1
RECRDF1: BXB1 + BXB1 RDF
REC2: TP901
RECRDF2: TP901 + TP901 RDF
RECS RFP: site1A + site2A + RFP+GFP rev + site1B + site2B
Assembly Flowchart
The assembly flowchart is as follows:
The parts will be replicated in a high copy plasmid, but a low-copy plasmid was chosen for characterization based on the importance of reducing the noise associated with having multiple copies of the fluorescent proteins’ sequence to be recombined. E. coli will be our chassis because of the previously executed recombinase characterization and counter circuits implementations. Strain BL21 was chosen because of its notable productivity in recombinant protein production and low protease activity (Kim et al., 2017)). For the assembly digestion, ligation, chemically competent cells production, and chemical transformation and colony PCR methodologies will be executed. You can find these in Protocols.
For the fluorescence characterization we propose four different approaches:
Plate Reader
Characterization of fluorescent protein expression via plate reader is a very common approach as shown by Beal et al. (2021). The main advantage is that even when our different constructs are designed to give us different pieces of information they can all be characterized at the same time, given that this is a high throughput methodology that allows bulk measurements (Fedorec et al., 2020), by simply inducing with arabinose and setting up the plate reader to the right wavelength according to the fluorescent protein of interest. The fact that even three different wavelengths could be processed at a time was a great advantage, since our plan consisted of inducing our counter circuit and continuously measuring the fluorescence of the three selected proteins, which, if working correctly, should be expressed sequentially as shown in description. This would also allow us to try out different arabinose concentration and induction times in order to normalize the pulse needed by our counter in order to count a single time. In the case of the pnrd promoter, which is endogenously induced, the plate reader should be able to capture the different expression peaks. Finally, the recombination rate of our recombinases can be easily extracted from this experimental procedure.
Flow Cytometry
Flow cytometry is also a very usual procedure to measure fluorescence (Beal et al., 2021), and, as with plate reading, behaves with the versatility needed to characterize our different constructs. This technique also presents the added bonus of segregating each specific cell by its expression phenotype (McKinnon, 2018), which allows the “detailed analysis of complex populations in short periods of time” (Adan et al., 2017). This technique will grant a much more accurate reading of our circuit’s behavior and probability profile, even allowing to directly correlate the obtained results with the Markov modelling of our counter circuit.
Live Cell Imaging
Finally, live cell imaging is proposed to characterize our counter circuit in a visual manner. Direct observation of fluorescence through microscopy can be very effective to determine live changes on a single cells’ behavior, as shown by Howell et al. (2017), which is what we would expect from our counter circuit.
DNA sequencing
DNA sequencing was suggested to us by several of the experts we interviewed, since it’s a direct outlook on the circuit’s state, compared to the indirect measurement obtained by fluorescence readings. For this approach the culture is to be induced and samples taken at different timepoints. After this a plasmid-DNA extraction and PCR amplification of the specific sequence should be performed. The samples would then be sent out to sequence and the results analyzed for qualitative information to be obtained.
Data Analysis
Both plate reading and flow cytometry are quantitative measurements that allow standardization via calibration and their execution can be designed in order to obtain comparable, replicable units (Beal et al., 2018). From these experiments we expect to obtain a behavioral pattern (counter circuit characterization) along with quantitative fluorescence measurements in which generation and degradation of the different fluorescent proteins are evident and allow the procurement of data for our mathematical model (overcount & recombinase characterization). The information obtained from live cell imaging will be used as qualitative visual characterization for our counter circuit. Nonetheless, arabinose concentration and elapsed time will be measured, allowing some data to characterize the induction system of our circuit. The pnrd promoter’s behavior will also be assessed by this mechanism.
01
Metabolic burden of our circuit, as implemented by https://2019.igem.org/Team:Austin_UTexas , who didn’t address recombinases in their study and haven’t established whether the total burden is equal to the sum of each individual part’s burden.
02
Anderson promoter characterization via standardized measurement in order to use as reference input for our promoter strength prediction tool.
03
Measurement of synthesis and degradation constants for recombinases, along transcription, translation and degradation rates for these proteins to allow an improved ODE’s model.
04
Evolutionary stability assessment (similarly to Fernández-Rodríguez et al. 2015).
Also, thought was put into possible characterization staging for the implementation of our project.
01
In vitro behavioral study
Studying the behavior of the engineered organism in similar conditions to the ones it will be subjected to.
02
Specific assays directed towards the gain of critical knowledge
Robustness, escape rate, optimal conditions, effectiveness of the desired function, etc.
03
In vitro interaction study
Understanding the interactions between the organism and some of the key organisms in the environment’s microbiome.
04
In vivo (enclosed) interaction study
Looking for the effect the engineered organism has in the environment it’s intended to target under controlled conditions.
05
In vivo interaction study (open, semi - controlled)
Observing the behavior of the engineered organisms and its repercussions in a semi-controlled context (i.e: waste-water treatment).
06
Small in situ study
Direct application of the organism for its intended purpose, small in scale, homogeneous conditions, simple interactions and as isolated as possible.
07
Large in situ study
Same as before, but testing different areas/conditions which are larger in scale, more diverse or complex.
08
Final application
*It is important to take into consideration that each of these stages might require separate approval by regulatory agencies. Experimental design will be key for the correct planning and execution of these phases, as well as for the obtaining of correct and valid data.
Molecular Biology
We took a record of every lab work day to have a register of the process and results. Each day of work, register the experiment, person in charge, a short description and results. In every description is stated the protocol used to achieve the goal of each specific experiment. Those protocols are punctually described in Protocols
The composite constructs created for our characterization were added to the repository, along with a DNA Ligase, the only part completely missing for the fulfillment of our suicide circuit design.
| Code | Name/description |
|---|---|
| BBa_K3917004 | DNA
Ligase 30 from Bacteriophage T4 |
| BBa_K3917001 | Autonomous genetic counter that tracks the number of times an Arabinose impulse is supplemented |
| BBa_K3917003 | Module to prevent over-count of a genetic autonomous counter. |
Graphic visualization of composite parts:
Counter circuit: {BBa_K3917001}
Overcount preventions system: {BBa_K3917003}
Due to the pandemic and the difficulties to access the laboratory, team members transportation, capacity of the lab and other external setbacks as delays in customs and shipment, the characterization of the composite parts in the laboratory was not possible. Nevertheless we made some in silico characterizations of the protein structure model and protein motif predictions for each protein of our composite parts BBa_K3917001 (Counter) and BBa_K3917003 (Module to prevent Overcount). The results of those in silico characterization are in the following documents:
