iGEM 2024 — Team aix-marseille


****BAC'ATTACK** ENGINEERING**

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* [Main Goal](#content1)
* [Design - Proof of Concept](#content2)
* [Build](#content3)
* [Test](#content4)
* [Learn](#content5)

# Main Goal

The goal of our METALE system, represented by part BBa\_K5422013, is to enable the controlled production of a modified Cyt2Aa1 toxin in the cytoplasm. Cyt2Aa1, a toxin naturally produced by Bacillus thuringiensis, is known for forming pores in the cell membranes of the intestinal barrier of insects like aphids, leading to their death. In our system, Cyt2Aa1 expression is driven by a strong ribosome binding site (RBS), ensuring high levels of toxin production.
At the same time, a weaker RBS regulates the production of T4 endolysin and T4 holin. When holin reaches a critical threshold, it induces pore formation in the bacterial membrane, allowing endolysin to penetrate and degrade peptidoglycan, ultimately leading to bacterial lysis and the release of large amounts of Cyt2Aa1. This results in aphid mortality. To fine-tune this mechanism, we plan to modify the RBS controlling the translation of T4 endolysin and T4 holin through site-directed mutagenesis. A library of 10 RBS variants will be generated to modulate their expression, using primers specifically designed to help select the most suitable mutant for our system.
![schéma romain](https://static.igem.wiki/teams/5422/e1.png)

# Design - Proof of Concept

![schéma romain](https://static.igem.wiki/teams/5422/e2.png)
Before creating the final BBa\_K5422013 part, we decided to build a proof of concept to validate our ideas using easily measurable genes. Our design involved linking various components together: a strong constitutive promoter, a strong RBS, GFP, another strong RBS, mCherry, and a terminator. While designing this sequence, we optimized codons for efficient expression in E. coli and ensured compatibility with BioBrick and Type IIS assembly standards. We also made adjustments to avoid secondary structures that could interfere with gene synthesis. These changes included synonymous codon substitutions to preserve the amino acid sequence while optimizing for synthesis and expression efficiency. Additionally, we designed the 10 sets of primers for performing RBS mutagenesis, which would be used based on fluorescence microscopy results. It was important to determine whether the length of the mRNA would affect translation levels, potentially making mutagenesis unnecessary.

# Build

To construct our system, we assembled the different fragments using the Gibson Assembly kit from New England Biolabs. PCR-directed mutagenesis was then used to create the 10 RBS mutants. After transforming E. coli DH5α with our constructs, 8 of the 10 mutants were confirmed to be correct.

These 8 were purified and sent for sequencing to verify whether the mutagenesis was successful.

# Test

Fluorescence microscopy showed strong expression levels of both GFP and mCherry before mutagenesis. Consequently, we proceeded with the mutagenesis experiments. The goal was to reduce mCherry expression relative to GFP by modifying the mCherry RBS.

![schéma romain](https://static.igem.wiki/teams/5422/e3.png)

 However, fluorescence readings revealed consistently low levels of GFP and almost no mCherry expression across all strains, including the wild type. This indicates that the RBS modifications might have impacted mCherry mRNA stability or translation efficiency.
Even though a strong promoter was used, the near-zero levels of mCherry suggest issues with either the promoter or the translation initiation process. Interestingly, prior microscopy (see Results) had shown co-expression of both proteins, but spectrophotometric analysis failed to detect mCherry, pointing to discrepancies between the two experimental approaches. Further experiments, such as testing different RBS designs or bacterial strains, will be required to resolve these issues and optimize the system.

# Learn

The spectrophotometric data were not usable and failed to validate the ideas proposed during the design phase. Nevertheless, fluorescence microscopy before mutagenesis clearly showed strong expression of both fluorescent proteins in the unmutated clone, indicating that the initial design was functional to some extent. These insights will guide the next iteration of the engineering cycle, particularly in refining the RBS design and addressing translation issues.

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Experiments

*This section details all the equipment used as well as the steps and protocols for each experiment in each of the three phases of the project.
Due to time constraints, some planned and prepared experiments could not be carried out; they are also detailed to demonstrate the thought process behind the entire project.*

## PHASE I: Cloning Experiments

##### A. Inventory of Primers and Genes

![](https://static.igem.wiki/teams/5422/experiments-table-1.png)

![](https://static.igem.wiki/teams/5422/experiments-table-2.png)

##### B. List of Experiments Performed

* Gene Amplification
  + PCR
  + Electrophoresis
* Fragment Purification
  + In liquid
  + On gel
* Vector Digestion by Restriction Enzymes
  + pBAD-hisC digest with HinIII and NcoI
  + pOK12 digest with HindIII and XhoI
* Cloning
  + SLIC
  + Gibson
* Plasmid Insertion into E. coli DH5α
  + Super competent bacteria
  + Transformation
* Clone Verification
  + Colony PCR
  + Restriction profile
  + Sequencing

##### Cloning diagrams

![](https://static.igem.wiki/teams/5422/diagram1.png)

![](https://static.igem.wiki/teams/5422/diagram2.png)

![](https://static.igem.wiki/teams/5422/diagram3.png)

![](https://static.igem.wiki/teams/5422/diagram4.png)

![](https://static.igem.wiki/teams/5422/diagram5.png)

![](https://static.igem.wiki/teams/5422/diagram6.png)

## PHASE II: Characterization of the Inducible Promoter and the VIRBAC Receptor

##### A. Experiments Performed

* Fluorescence Characterization with Xylose Induction
  + Xylose gradient
  + Spectrofluorimetry
* Receptor Production with Xylose Induction
  + Xylose gradient
  + Western blot
* Receptor Production with Xylose Induction and Glucose Inhibitor
  + Xylose induction
  + Glucose gradient
  + Western blot
* Membrane Insertion Verification
  + Xylose induction
  + Cellular fractionation
  + Western blot

##### B. Planned (Not Performed) Experiments

* Fluorescence Characterization with Xylose Induction and Glucose Inhibitor
  + Xylose induction
  + Glucose gradient
  + Spectrofluorimetry
* Membrane Fluorescence
  + Spheroplasts
  + Immunolabeling
* Measurement of Receptor Dimerization
  + Caffeine gradient
  + Spectrofluorimetry

## PHASE III: Characterization of the Double Level Expression System METALE

##### A. Inventory of Primers Used

![](https://static.igem.wiki/teams/5422/experiments-table-3.png)

##### B. List of Experiments Performed

* Fluorescence Characterization by Microscopy (Constitutive Promoter)
  + Sample preparation
* Fluorescence Characterization with TECAN (Constitutive Promoter)
  + Spectrofluorimetry
* Mutagenesis of RBS (Constitutive Promoter)
  + Quick change PCR reaction
  + Transformation
  + Screening
  + Mini prep
  + Sequencing
* Fluorescence Characterization of Mutants (Constitutive Promoter)
  + Spectrofluorimetry

##### C. Planned (Not Performed) Experiments

* Fluorescence Characterization by Microscopy (Inducible Promoter)
  + Caffeine induction
  + Sample preparation
* Fluorescence Characterization with TECAN (Inducible Promoter)
  + Caffeine gradient
  + Spectrofluorimetry
* Mutagenesis of RBS (Inducible Promoter)
  + Quick change PCR reaction
  + Transformation
  + Screening
  + Mini prep
  + Sequencing
* Fluorescence Characterization of Mutants (Inducible Promoter)
  + Caffeine gradient
  + Spectrofluorimetry
* Verification of Protein Production (Inducible Promoter)
  + Caffeine gradient
  + Western bloc
* Visualization of Bacterial Lysis
  + Transformation (cytoplasmic fluorescent protein)
  + Caffeine gradient
  + Video of bacteria

****BAC'ATTACK** RESULTS**

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* [PHASE I : Cloning Experiments](#content1)
* [PHASE II : Characterization of the Inducible Promoter and VIRBAC](#content2)
* [PHASE III : Characterization of the METALE Double-Level Expression System](#content3)

# PHASE I : Cloning Experiments

In the initial stages of our cloning experiments, we encountered an unexpected issue: entire pools of colony PCRs were either empty or produced no amplification products (very rare). We hypothesized that the assembly might have been hindered by contamination from unwanted fragments, potentially interfering with the reaction. To address this, we implemented stricter PCR conditions for the fragments production and proceeded with gel purification to ensure only the correct fragments were present.

Example of colony PCR results from the PcGMc cloning.
![schéma romain](https://static.igem.wiki/teams/5422/r1.png)

 However, despite these precautions, the next round of results revealed that many of the clones contained inserts of incorrect sizes, with some even showing significant deletions. This led us to suspect that something more complex was at play that our constructs were toxic even though we didn’t see such things during our research.

Examples of colony PCR results for the XARcaf, XARctl, and XAG clonings (from left to right).
![schéma romain](https://static.igem.wiki/teams/5422/r2.png)

After nearly two months of troubleshooting, and having tested almost every conceivable variable, we tested two cloning methods (SLIC and Gibson HiFi assembly). Unfortunately, these attempts yielded similar results so we decided to try one last approach as the end of the internship drew near. We revisited our initial clones, where colony PCR had seemingly failed, and performed PCR colony screening using Q5 high-fidelity polymerase (red circles on the first image). This decision turned out to be a turning point. The results were markedly improved, with clear and correctly sized bands appearing more consistently, suggesting that the fidelity of the Q5 polymerase was crucial in resolving the earlier issues.Following these improvements, we successfully obtained 4 out of the 7 initially planned constructs:

Colony PCR results of XAG using Q5 polymerase.

 ![schéma romain](https://static.igem.wiki/teams/5422/r3.png)

Colony PCR results of XARcaf using Q5 polymerase.
![schéma romain](https://static.igem.wiki/teams/5422/r4.png)

Colony PCR results of XAGctrl using Q5 polymerase.
![schéma romain](https://static.igem.wiki/teams/5422/r5.png)

Colony PCR results of PcGMc using Q5 polymerase.
![schéma romain](https://static.igem.wiki/teams/5422/r6.png)

# PHASE II : Characterization of the Inducible Promoter and the VIRBAC Receptor

**Characterization of the XylA promoter**
![schéma romain](https://static.igem.wiki/teams/5422/r7.png)
Analysis of Pxyl promoter induction by xylose revealed significant differences between conditions with and without glucose. The figure shows that in the absence of glucose, increasing the concentration of xylose leads to an increase in fluorescence. The KM parameter, estimated at 33.2 ± 18.5 mM, indicates the concentration of xylose at which half of the maximum fluorescence is observed.

**Characterization of the caffeine receptor**

We conducted a western blot to evaluate the functionality of the PxylA-RBS-caffeine receptor construct, specifically focusing on caffeine production. To determine the optimal xylose concentration for promoter induction and gene expression, we prepared a gradient of xylose concentrations (1%, 2%, 3%, 4%, 5%, and 10%), along with an uninduced control. However, no protein was detected via western blot, which raised concerns about potential issues with expression or translation.

Given the deletions observed in previous cloning attempts, we hypothesized that the receptor protein might have toxic effects on the cells, potentially due to promoter leakage. To investigate this, we monitored the optical density (OD) during bacterial growth in the presence of xylose, as shown in the graph. The growth curves revealed no adverse effects on bacterial growth, ruling out the possibility of toxicity from the caffeine receptor.

 ![schéma romain](https://static.igem.wiki/teams/5422/r8.png)

Following the western blot results, we sent the PxylA-RBS-caffeine receptor construct for sequencing. Sequencing revealed a premature stop codon, or nonsense mutation, which explained the lack of protein detection. Unfortunately, due to time constraints, we were unable to reclone the construct and attempt a new PxylA-RBS-caffeine receptor build to achieve caffeine receptor production.

**Characterization of the control receptor**

We performed a western blot to assess the functionality of the PxylA-RBS-control receptor construct by inducing its expression using varying concentrations of xylose, similar to the experiment conducted with the caffeine receptor. The range of xylose concentrations used included 1%, 2%, 3%, 4%, 5%, and 10%, along with a non-induced control (0% xylose). The results showed the presence of the control receptor protein at the expected size of approximately 35.5 kDa. However, we observed no increase in protein production despite the increasing concentrations of xylose. This suggested that these xylose concentrations might be saturating the inducible promoter. Once this threshold is reached, additional xylose does not lead to further protein expression, indicating that the promoter has a limit beyond which it no longer induces increased protein production.To test this hypothesis, we conducted another experiment using a lower range of xylose concentrations: 0% xylose, 0.05% xylose, 0.2% xylose, 0.4% xylose, 0.6% xylose, and 0.8% xylose. The results of the second western blot confirmed an increase in control receptor protein production at these lower xylose concentrations. This validated our hypothesis that xylose concentrations above 1% indeed saturate the promoter, preventing further induction of protein expression.

 ![schéma romain](https://static.igem.wiki/teams/5422/r9.png)

As with the caffeine receptor, we conducted a toxicity test by measuring the optical density (OD) of each culture in the presence of xylose. Measurements were taken every hour over a 7-hour growth period. The results were consistent with those observed for the caffeine receptor, indicating that no toxic effects on cell growth were detected, even in the presence of xylose. These findings confirm that our constructs do not negatively affect the bacterial cells' growth under these experimental conditions.

 ![schéma romain](https://static.igem.wiki/teams/5422/r10.png)

Due to time constraints, we would have liked to test the induction of our promoters in the presence of xylose as well as another sugar, such as glucose, to observe its effects on the inducible promoter. This would have allowed us to evaluate the impact of catabolite control on protein production, providing insight into how different carbon sources affect promoter activity and gene expression.

# PHASE III : Characterization of the METALE Double-Level Expression System

**Characterization of the PcGMc construct**
![schéma romain](https://static.igem.wiki/teams/5422/r11.png)

For this biological sample, six different fields of view (F.O.V) were captured to ensure a comprehensive representation of the entire sample, avoiding potential biases that could arise from examining only one region, which might be atypical or abnormal. By selecting two distinct fields of view (F.O.V 1 and F.O.V 2), we ensured that our observations were representative of the entire sample and not an isolated occurrence.

To present our results, we selected two fields of view from the six, demonstrating that our bacteria simultaneously produce the fluorescent proteins GFP and mCherry. The figure demonstrates the co-localization of GFP and mCherry fluorescent signals within the bacterial cells. This co-expression is visible in the "Merged" images (c and f), where the overlap between GFP's green fluorescence and mCherry's red fluorescent results in yellow regions, confirming simultaneous expression of both proteins in the cells. As a result, our construct successfully expressed the GFP and mCherry genes.

Additionally, to standardize our results and minimize noise contamination, we reduced the brightness and contrast of GFP fluorescence using the software ImageJ 1.54g Java 1.8.0\_345 (64-bit) due to its overly strong intensity. Adjusting the brightness and contrast in the GFP channel prevented over-saturation, which could have obscured details or led to misinterpretation of the data. This highlights the importance of maintaining precise controls in fluorescence microscopy to ensure the accuracy and reliability of the results.

The consistent expression of both fluorescent markers across multiple fields of view further supports the conclusion that the genetic construct is stably expressed in the bacterial population, with minimal variation between cells.

 ![schéma romain](https://static.igem.wiki/teams/5422/r12.png)

 The table data were generated after performing site-directed mutagenesis on the ribosome binding site (RBS) of the mCherry gene, with the intent to reduce its expression relative to GFP. The goal was to achieve differential expression between the two proteins, with mCherry being expressed at lower levels. However, the results show consistently low GFP levels and exceptionally low or negative RFP values across all E. coli strains, including the non-mutated wild type (WT). The near-zero RFP values, especially in mutants such as M2 and M5, suggest that the RBS modifications may have significantly disrupted the stability of mCherry mRNA, its translation efficiency, or overall gene expression.

 In some mutants, GFP expression is still relatively close to the original construct (e.g., M3, M4, and M9), but others like M2 show very low GFP levels, indicating uneven effects of the mutations on GFP expression. The consistent lack of mCherry fluorescence suggests that the RBS mutations have had a more profound effect on its expression, possibly hindering translation initiation altogether.

Despite using a strong constitutive promoter, these data raise concerns about its effectiveness under these conditions. It seems that either the promoter is malfunctioning or the RBS mutations have had unintended consequences on mCherry production. Even the wild-type strain, which should show both GFP and mCherry expression, failed to produce detectable mCherry fluorescence, reinforcing the possibility of systemic issues with either the promoter or translation initiation.

Interestingly, prior fluorescence microscopy experiments showed clear co-expression of GFP and mCherry in these bacterial cells. However, the spectrophotometric data tell a different story, with no detectable mCherry across any strains. Both sets of data were collected from the same strains under nearly identical conditions, with only a short delay due to sequencing. This discrepancy between the microscopy results and the spectrophotometric readings suggests potential experimental inconsistencies or differences in sensitivity between the methods used.

To account for these unexpected results, several factors must be considered. Experimental variability, such as slight measurement inaccuracies or fluctuations in culture conditions, could have played a role. In complex systems involving multiple variables—like promoter strength, RBS efficiency, and protein folding—even minor differences can significantly impact gene expression.

Another possibility is that the RBS mutations affected mCherry mRNA stability. If these mutations caused structural changes in the mRNA, they could have made it more prone to degradation or less efficient in initiating translation, thereby reducing or eliminating mCherry protein production. Such effects could occur even if transcription proceeded normally, and this would explain why no mCherry fluorescence was observed.

Furthermore, interactions between the bacterial host and the synthetic construct might have contributed to the lack of mCherry expression. The host cells may have regulatory mechanisms that interfere with the expression of mCherry, particularly if the modified RBS reduced the efficiency of ribosome binding or mRNA processing.

Complementary investigation is necessary to clarify these findings. Testing alternative RBS designs or using different bacterial strains may provide insights into the lack of mCherry expression. Altering the promoter or modifying other regulatory elements could also help resolve the expression issues. As is often the case in synthetic biology, iterative design cycles, including testing and optimization, are critical for overcoming unexpected challenges and achieving the desired system functionality.

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