Engineering success

iGEM gave us the chance to be part of an engineering cycle from start to finish. We learnt what it means to plan, design, build and test our test system and how important it is to integrate feedback from experts during every step.

Following the principles of the engineering cycle was completely new to us as we did not previously have the opportunity to plan a project from start to finish all by ourselves. However, we quickly realized how much easier it was to visualize the progress, celebrating successes and handling hurdles of our project. We also discovered the importance of consulting experts at each stage, a key ingredient for success

Figure 1: The iGEM Engineering cycle which we used as a template.

Engineering Cycle: Construct Engineering

The objective of our project was to build a cell free system that enables quantification of Lithium in samples with a riboswitch-reporter system. Here, you can learn about how we achieved our objective by following the design, build, test, learn cycle.

Stage 1

In this stage, we developed the first prototype of our construct with the fluorescent reporters and different versions of the T7 Promoter-Riboswitch system (1-4G, 1-4M). You can find an overview over the constructs on the results page. After in silico design and ordering of the DNA, we started with the first round of Golden Gate cloning. After verifying the cloning success by sequencing and transformation into E. coli, we started liquid culture assays. During literature research, we found out that bacteria very efficiently import lithium ions at pH 9, which is why we adjusted the main culture pH to this value (2). However, we realized in the building step that we needed to optimize the assay conditions for our bacteria: We could not detect any signal in our measurements after adding 50 mM LiCl to the growth media in the testing stage. We suspected that this was caused by insufficient bacteria growth at the suggested pH 9.

Furthermore, we had a negative control in our original test setup (riboswitch-reporter construct without lithium) but no positive control. Because of the missing positive control, we could not evaluate with final certainty whether the problems with the measurement were also caused by a problem in our DNA construct.

We also took into account that the missing signal could also be explained by the fluorescence reporter not being sensitive enough. Therefore, we decided to research alternative, more sensitive reporters.

Three things we learned during stage one that we implemented in stage two:

  1. Besides a negative control, a positive control is very helpful for result analysis. A signal from the positive control would indicate that the test setup is working. We therefore decided to create constructs that expresses the reporters under a T7 Promoter but without riboswitch as our positive controls
  2. The optimal growth conditions are vital for bacteria based assays and can greatly vary even between different strains of the same bacteria. Because of this, we planned pH assays in stage 2 to determine optimal growth conditions
  3. We tested the sfGFP and mScarlet-I3 constructs with the ribsowitch variants 1-4 in this stage because we thought that fluorescent reporters would be the best reporters for our project as they are easy to work with. However, we hypothesized that we did not get a good signal in this stage because the fluorescent reporters are not sensitive enough, hence we started looking into other options.

Figure 2: The First Stage of the DBTL-Cycle.

Stage 2

Stage one of the engineering cycle showed us that we had many adjustments to make. It is for this reason we reached out to an expert in the field of Synthetic RNA Biology: Prof. Suess. She suggested that we try the luminescent reporter NanoLuc which is a lot more sensitive than fluorescent reporters. We also designed the positive control consisting of only the T7 promoter and the respective reporter, which enabled us to compare signal intensities not only to a negative control but also to the signal without riboswitch regulation (see Parts BBa K4654021, BBa K4654022, BBa K4654023).

After everything was ordered and cloned, we proceeded to the build step. We tested pH 7.5, pH 8 and pH 8.5 in the liquid culture assays as well as LB medium, LB low sodium medium and TB medium because we realized that the salt concentration in the media could affect the measurements. The results showed that we had the best growth conditions at pH 7.5 (Fig. 4) and the measurements in different media showed us that we received the highest reporter signals when bacteria were grown in LB low sodium medium (Figure 5). Because of this, follow-up experiments were done under these optimized conditions. In the learn stage, we drew conclusions from our results that we could implement in stage three: We discussed that the R1-Nanoluc construct was the most suitable for our project because it repeatedly delivered the best signals (see results). But we also discussed that to proof our concept, we needed to start implementing this construct in a cell free environment.

Figure 3: Influence of pH on Bacteria Growth This figure shows bacteria growth (OD600) in LB media at different pH. E. coli KRX transformed with constructs 1M with induced T7 Promoter, 5M (PC) and 1M without induced T7 Promoter (NC) were tested

Figure 4: sfGFP Expression in Different Media The figure shows sfGFP expression for E. coli KRX bacteria transformed with the 5G construct over a timeframe of 330 Minutes. Bacteria were grown overnight in respective media as pre cultures. The next day, main cultures were inoculated and grown until OD600 0.1, then 0.1% rhamnose was added to induce sfGFP expression. sfGFP expression was measured with a Tecan Spark Plate Reader. Normalization of sfGFP expression values were done by dividing the RFU by the respective OD600 values.

Two things we learned during stage two that we implemented in stage three:

  1. Our most promising candidate for our test system was construct 1N (BBa_K4654018). This realization enabled us to disregard most other constructs and save time and resources during the third iteration of the design build test learn cycle
  2. We realized that we had reached the step where we could start implementing the cell-free system

Figure 5: The Second Stage of the DBTL-Cycle.

Stage 3

Stage three was all about taking our test system from a mere proof of concept to a cell-free solution that could be used outside of a laboratory. We started designing the cell-free system with input from experts on Bipolar and lithium medications because we envision our end product to be as user friendly as possible. However, when we entered the build phase, we realized we had to step away from the idea to create our own cell free system as we were running out of time and resources. Since we still wanted to show that we were able to proof our concept, we decided to use a commercial cell-free kit by biotechrabbit. This way, we could still enter the test phase which showed positive results for the constructs with nanoLuc and sfGFP, thereby proving that we were able to create a riboswitch based system for the detection and possible quantification of Lithium that is also functional in a cell-free environment (Figure 6). However, due to limited resources, we could not work with biological replicates, which is why the assay we used to test different lithium concentrations to determine the correlation between lithium concentration and reporter expression did not work as expected (Figure 6).

Figure 6: Expression of NanoLuc under the Control of a Lithium Sensitive Riboswitch (Construct 1N) in a Cell Free Environment. The obvious increase of signal between 0 mM and the reactions with LiCl show that the construct also works in a cell-free environment. However, it is also clearly visible that the quantification did not work as one would expect the signal to get stronger with a higher LiCl concentration. This led us to the conclusion that we need to repeat the experiment with replicates.

With these results in mind, we entered our final learn step of our project:

We learned that in further experiments, we would need to work with at last 3 biological replicates of each test conditions to get a meaningful result in a cell-free setup

Figure 7: The Third Stage of the DBTL-Cycle.

Sources
  1. https://technology.igem.org/engineering/introduction(last opened on 10th Oct 2023, 2:43 pm)
  2. White, N., Sadeeshkumar, H., Sun, A. et al. Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes. Sci Rep 12, 19145 (2022). https://doi.org/10.1038/s41598-022-20695-6