iGEM 2022 — Team goethe-frankfurt


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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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### **How does our design cycle look like?**

### **Introduction**

With the goal to engineer *E. coli* strains that can both detect and absorb free Cadmium from native water sources, we first had to test the validity of our concept on a laboratory scale. Here, we envisioned two separate systems relying of the overexpression of several endogenous and exogenous proteins that would work in tandem to let the host perform these tasks.

As the expression of let alone one but up to four proteins was expected to put a strain on *E. coli*, we had to ensure that our engineering approach was up to the task.

1. **Design:** Both the envisioned system for cadmium detection via chemotaxis as well as its bioremediation through increased import and sequestration required the co-expression of up to four endogenous and exogenous proteins. As the production of such considerable amounts of protein could perturb the hosts proteomic equilibrium as well as cause formation of inclusion bodies, we had to carefully design each proteins expression cassette.
2. **Build:** To achieve the co-expression of our proteins of interest from a single plasmid, we used a GoldenGate cloning approach based on the pOdd/pEven Loop vector system. Here, expression of single proteins was possible via transformation with Level1 constructs, whereas transformation with Level2 constructs allowed co-expression of these proteins.
3. **Test:** We conducted a variety of gain-of function tests to assess the functionality of designed protein expression cassettes both alone and during co-expression.
4. **Learn:** Testing the functionality of our proteins of interest revealed different levels of functionality. If given more time in the laboratory, additional engineering cycles would have focused on designing and implementing protein cassettes with alternative promoter strengths. Also, based on conclusions during the testing phase, more inducible instead of constitutive promoters would have been implemented to toggle protein expression more precisely.

### **Cycle 1**

##### 1.1 Design

The purpose of our design was to achieve constitutive expression of several proteins of interest that would together achieve one of two goals: the detection of cadmium through specific chemotaxis as well as cadmium uptake from the environment through its increased import into the cytoplasm as well as subsequent complexation through one of two storage proteins. For both systems to work as intended we had to ensure the respective co-expression of four proteins of interest. To this end, we planned a cloning regiment in which we would first try to insert the gene for each of our proteins of interest into a Level1 construct under the control of one of four constitutive promoters each (J23100, J23101, J23114, J23119). Given the varying strength of these promotors, we had hoped to be able to test different combinations of these expression cassettes in Level2 constructs to toggle co-expression in the host. Keeping in mind the problem of homologous recombination when it comes to transforming Level2 constructs, we initially also planned to combine each protein’s CDS with a specific set of ribosomal entry sites and terminators. Here, we paid special attention to ensure that in the final Level2 constructs, there would be no sequential repeats between the different expression cassettes. An example for a Level1 as well as Level2 construct is shown in Fig. 1.

![Example of a Level1 and Level 2 construct.](https://static.igem.wiki/teams/4285/wiki/engineering/engineering-success-igem22-fig1.png)

***Fig.1 Example of a Level1 and Level 2 construct.** Level 1 construct (left) contains a full functional cassette for the expression of a single construct of interest. The cassette is comprised of a promotor (J23100), a RBS (B0034) the CDS (CysE) and a terminator (J61084). Level2 constructs (right) harbor up to four fully functional expression cassettes, in this case those for CysP and CysE.*

##### 1.2 Build

To obtain our constructs of interest, we relied on a GoldenGate cloning strategy based on the pODD/pEVEN Loop vector system. At first, we set out to insert the synthesized CDS directly into a Level1 construct (pOdd1, pODD2, pOdd3, pOdd4). This was possible as the synthesized sequences were designed with overhangs containing recognition sites for compatible type IIS restriction enzymes. Initial cloning approaches include those synthesize sequences that already contained a RBS (B0030, B0031, B0032, B0034) as well as a terminator (B1001, B1002, B1005, B10015, J61084) in addition to the CDS. We then combined these synthesized sequences with one of four promoters (J23100, J231001, J231014 & J231019) each and inserted them into Level1 backbones. While this approach followed our initial plan to assemble four versions of each protein’s expression cassette, all with specific RBS-terminator pairs and each with one of four promotors, we were not able to obtain all our desired constructs this way.

### **Cycle 2**

##### 2.1 Design

As our initial cloning attempt using synthesized sequences comprised of not only a CDS but also an RBS as well as a terminator did not yield all our desired constructs, we went back to the drawing board to re-evaluate our approach. We therefore decided to instead try and assemble the missing constructs by combining synthesized fragments only containing the CDS with separate Golden Gate compatible RBS and terminator sequences. In addition, we also decided to try and incorporate inducible instead of constitutive promotors for some of our failed constructs, as we argued that constitutive expression of some of our proteins of interest might impede the growth of our production strains.

##### 2.2 Build

We set out to assemble those Level1 constructs still missing for Level2 plasmid assembly using synthesized sequences only containing the respective CDS flanked by compatible overhangs. Due to availability issues, all these CDS had to be paired with the same combination of RBS (BCD2) and terminator (EZK9600) sequences. We also introduced inducible promoters (LacZ & T7) into some constructs we hypothesized to otherwise affect the growth of production strains. With this combined approach, we were then able to generate all missing Level1 constructs from our first engineering cycle.

All obtained Level1 constructs of interest were validified using a combination of blue-white-screening, diagnostic digests, colony PCR as well as commercial sanger sequencing. Assembly of Level2 constructs saw the insertion of up to four Level1 expression cassettes into Level2 backbones (pEven1, pEven2, pEven3, pEven4). While we were able to construct a Level2 construct enabling the co-expression of all proteins necessary for our chemotaxis-based detection concept, we were not able do the same for our bioremediation approach. Instead, we opted to employ a fully synthesized Level2 plasmid to test the feasibility of our bioremediation concept.

##### 2.3 Test

To test both our cadmium detection as well as bioremediation approach, we set out to test the functionality of introduced proteins of interest both expressed alone as well as together. Due to the limited timeframe during which we had to conduct our project, we decided to limit our testing to gain-of function studies.

For our detection approach, this meant to show that chemotaxis-deficient *E. coli* UU3330 (kindly provided by Prof. Parkinson, University of Utah) would exhibit swarming behavior towards a source of Cadmium. To this end, we conducted Drop-plate assays where we introduced samples of different chemicals that act as natural chemoattractant onto LB-Agar plates containing homogenously distributed *E. coli* strains. Here, we compared the swarming behavior of an *E. coli* Wild-Type (*E. coli* BL21) with those of the chemotaxis-deficient strain (*E. coli* UU3330) on its own and transformed with our Level2 construct (*E. coli* UU330-CHIU) (Fig.2). Here, we ran into several problems.

First, while we saw the formation of rings around our Cadmium chloride sample on the UU330-CHIU plate, we saw similar patterns in the UU330-Wildtype and the BL21-Wildtype. Therefore, we could not show specific chemotaxis towards cadmium based on the expression of our proteins of interest. Also, we saw that both the Bl21-Wildtype as well as the UU33 wildtype showed swarming behavior towards a variety of bivalent anions, which were include in the test to exclude non-specificity of our intended swarming behavior towards cadmium. In summary, we were not able to show specific chemotaxis towards a source of cadmium in an otherwise chemotaxis-deficient *E. coli* strain.

![Drop-Blot assay to compare swarming behavior of E. coli Wild-Type with chemotaxis-deficient E. coli UU330 transformed with our Level2 chemotaxis-construct.](https://static.igem.wiki/teams/4285/wiki/engineering/engineering-success-igem22-fig2.png)

***Fig.2 Drop-Blot assay to compare swarming behavior of *E. coli* Wild-Type with chemotaxis-deficient *E. coli* UU330 transformed with our Level2 chemotaxis-construct.** Shown are several Agarose plates containing homogenously distributed *E. coli* in response to several chemicals to assess chemotaxis behavior. No major difference can be observed between an *E. coli* BL21 Wild-Type and chemotaxis-deficient *E. coli* UU3330-WT. No alterations regarding the attraction towards Cadmium Chloride can be observed, even in *E. coli* UU3330-CHIU, which was transformed with our Level2 Chemotaxis construct.*

###### 2.3.1 Cadmium Bioremediation

Simultaneously to our chemotaxis assays, we set out to validate the functionality of our Cadmium-Bioremediation approach. To achieve this, we defined four questions we wanted to answer.

1. Does the expression of components of our Bioremediation alone as well together improve the survival of *E. coli* under increasing cadmium concentrations?
2. Can we show an increased import of sulfate from the environment?
3. Can we show increased synthesis of Cysteine within our transformed *E. coli*?
4. Can we show increased uptake of Cadmium from the environment?

To answer the first of our questions aimed toward the survival behavior of *E. coli* both with and without our bioremediation constructs, we conducted plate-based survival assays (Fig.3). However, as clearly visible for all tested constructs as well as combination of constructs, we were not able show increased tolerance towards Cadmium in comparison to the Wild-Type.

![Survival assay comparing the resilience of both E.coli WT and different combinations of our Bioremediation constructs towards increasing Cadmium concentrations.](https://static.igem.wiki/teams/4285/wiki/engineering/engineering-success-igem22-fig3.png)

***Fig.3 Survival assay comparing the resilience of both *E. coli* WT and different combinations of our Bioremediation constructs towards increasing Cadmium concentrations.** Dilutions series *E. coli* Wild-Type as well as variants transformed with different combinations of constructs constituting our Bioremediation approach were applied onto LB-agar plates containing increasing amounts of Cadmium.*

Next, to assess whether our Bioremediation construct allows for an increased uptake of sulphate intended to assist endogenous synthesis of Cysteine necessary for the overproduction of cysteine-rich proteins, we performed a sulphate uptake assay (Fig. 4). Here, we monitored the sulphate concentration within the supernatant of samples taken from a liquid *E. coli* culture over time. However, we were not able to detect any significant differences in the sulphate uptake behavior between the *E. coli* Wild-Type and *E. coli* transformed with parts of our Bioremediation constructs.

![Sulphate uptake assay.](https://static.igem.wiki/teams/4285/wiki/engineering/engineering-success-igem22-fig4.png)

***Fig. 4 Sulphate uptake assay.** We compared the sulphate uptake assay between *E. coli* WT and *E. coli* transformed with several of our Bioremediation constructs by measuring the concentration of sulphate in the supernatant of a liquid culture of several hours. No significant difference in the uptake behavior could have been shown.*

While our testing phase plan next saw the assessment of cystine synthesis, we, again due to time issues, decided to directly test Cadmium-Uptake in a similar manner as we did for sulphate (Fig. 5).

![Cadmium uptake assay.](https://static.igem.wiki/teams/4285/wiki/engineering/engineering-success-igem22-fig5.png)

***Fig. 5 Cadmium uptake assay.** We compared the cadmium uptake assay between *E. coli* WT and *E. coli* transformed with several of our Bioremediation constructs by measuring the concentration of cadmium in the supernatant of a liquid culture of several hours.*

When comparing the concentration of cadmium in the supernatant of the *E. coli* WT culture to those of expressing we do see some interesting trends. First, the expression of both our cadmium storage proteins alone (AtPCS1, hMT2) does not seem to increase Cadmium uptake (Fig. 5.a.). Interestingly, expression of our cadmium importer (MntH) together with our Cysteine synthesis cassette also does not lead to increased Cadmium uptake. However, we can discern a trend in which increased cadmium uptake can be observed in *E. coli* expressing EcCARs (Fig. 5.b.).

##### 2.4 Learn

From our engineering cycle we have learned that most of our constructs to not yet show the functionality we intended for them. During our first engineering cycle we learned that constitutive expression of our constructs especially when being expressed with other constructs might be the leading cause for problems during functional implementation. Also, more effort needs to be put into analyzing and testing our constructs individually on the Level1 construct basis to characterize them more thoroughly. This could include the quantification of His-tagged versions of our proteins depending on different promoter strengths. This approach could also test whether transformation of Level2 constructs harboring several expression cassettes could lead to interference in the production rate of each construct. Overall, a more comprehensive analysis of our obtained construct and their behaviour once transformed into a host is necessary. Also, we have seen that the incorporation of inducible instead of constitutive promoters has in some cases proven to lead to better results when it comes to construct-amplification in production strains. One could therefore consider the design of Level2 constructs were proteins of interest having an adverse effect on host cell survival would be put under induction control.

To investigate swimming behavior of different *E. coli* strains, which has also been used in qualitative chemotaxis analysis, we used a chip-based assay utilizing µ-slide chemotaxis chamber (IBIDI GmbH, Martinsried, Germany). These chips were previously used to quantify chemotactic response towards chemoattractants. Unfortunately, we were not able to generate results which could have been analysed to determine swimming speeds at different Cd2+-concentrations.

We intended to adapt the migration speed of *E. coli* UU3330-CHIU to larger chips (µ-Slide I Luer0.2 and µ-slide VI0.4 chips, IBIDI GmbH, Martinsried, Germany) where we wanted to relate migration speed to different Cd2+ concentrations in a given sample. This could have been leading to an easy to use and fast method to give first hints on Cd2+ contamination in the field.

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