iGEM 2023 — Team bulgaria

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

Welcome to the vibrant world of our close-knit Synthetic Biology Team! While our core mission revolves around pioneering research in synthetic biology, we're not your typical laboratory-based scientists. We are a dynamic team that believes in the power of connection, knowledge sharing, and, most importantly, having fun while we do it!

This notebook is a portal into our journey, as we blend science, creativity, and social activism to make an impact. Here, we share our adventures beyond the lab, as we engage with fellow enthusiasts, ignite curiosity, and promote our field in a unique and enjoyable way.

# March

![](https://static.igem.wiki/teams/4855/wiki/notebook/1march.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/2march.svg)
❮
❯

# April

![](https://static.igem.wiki/teams/4855/wiki/notebook/1april.svg)
❮
❯

# May

![](https://static.igem.wiki/teams/4855/wiki/notebook/1may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/2may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/3may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/4may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/5may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/6may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/7may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/8may.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/9may.svg)
❮
❯

# June

![](https://static.igem.wiki/teams/4855/wiki/notebook/1june.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/2june.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/3june.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/4june.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/5june.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/6june.svg)
❮
❯

# July

![](https://static.igem.wiki/teams/4855/wiki/notebook/1july.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/2july.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/3july.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/4july.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/5july.svg)
❮
❯

# August

![](https://static.igem.wiki/teams/4855/wiki/notebook/6august.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/7august.svg)
❮
❯

# September

![](https://static.igem.wiki/teams/4855/wiki/notebook/8september.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/9september.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/10september.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/11september.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/12september.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/13september.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/14september.svg)
❮
❯

# October

![](https://static.igem.wiki/teams/4855/wiki/notebook/15october.svg)
![](https://static.igem.wiki/teams/4855/wiki/notebook/16october.svg)
❮
❯

* [March](#march)
* [April](#april)
* [May](#may)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#september)
* [October](#october)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

*We employed the Engineering Design Cycle as a systematic method to enhance our CADABRA enzyme cocktail's efficacy in eliminating antibiotics from wastewater during our iGEM odyssey in 2023. This approach encompassed a sequence of stages that provided us with valuable guidance throughout our project.*

**1) Identify the Problem:**

Analyzing the results from last year’s project, we realized that the VIM-2 metallo-β-lactamase (MBL)-based version of the CADABRA cocktail suffers from some limitations in its efficiency. As visualized in our urine decontamination test (https://2022.igem.wiki/bulgaria/proof-of-concept), the meropenem levels in this experiment were reduced from 750 ug/ml to approximately 0.4 ug/ml in 12 hours. **Our main goal for this year was to accomplish complete removal of the antibiotic.**

Furthermore, while investigating the problem of antibiotic pollution in Bulgaria, we came across data indicating a concerning prevalence of antibiotic-resistant bacteria in some significant rivers within our country. Notably, **the most pronounced levels of resistance were observed concerning the β-lactam antibiotic ampicillin, which is commonly employed in animal farming** (Tsvetanova, Z.; Najdenski, H. Antimicrobial Resistance of Heterotrophic Bacteria and *Enterobacteriaceae* Inhabiting an Anthropogenic-Affected River Stretch in Bulgaria. *Processes 2023, 11, 2792*. https://doi.org/10.3390/pr11092792)

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/1.png)

**Ampicillin**

**2) Research and Gather Information**

In our quest for the requisite information, we conducted extensive literature research and arranged several meetings with medical professionals, including specialists in medical microbiology, hematology, and clinical pharmacology. To gain deeper insights into the issue of antibiotic contamination in clinical settings, we also engaged in discussions with the head of the government agency responsible for hazardous hospital waste management.

**3) Brainstorm, Generate Ideas and Select the Best Solution**

As we contemplated methods to enhance the efficiency of carbapenem degradation, two potential opportunities emerged: the elevation of MBL enzyme production to yield a more concentrated cocktail or **the augmentation of MBL enzymatic activity**. Our choice was to concentrate our efforts on the latter option. In pursuit of improving MBL activity, our initial step was to assess whether any naturally occurring MBL enzyme displayed greater activity than VIM-2. Subsequently, our plan involved implementing directed evolution techniques to further boost its enzymatic efficiency.

In our endeavor to combat ampicillin contamination in the rivers, our initial strategy was to determine the prevalent form of this antibiotic's usage in animal farming. Subsequently, our plan  **assessing both the original CADABRA cocktail and the enhanced version to determine their efficacy in degrading this β-lactam antibiotic in aquatic environments.**

**4) Design and Prototype**

We designed two novel MBL coding sequences, one based on IMP-1 (Q79MP6), and the other derived from a modified version of NDM, incorporating the mutations found in variants 2 and 5. These sequences were synthesized as codon-optimized gBlock fragments by IDT. To simplify the cloning process, we included VF2 and VR primer annealing sites at both ends of the fragment, along with the BioBrick Prefix and Suffix sequences. Subsequently, we introduced these constructs into a vector featuring a strong promotor and medium RBS (BBa\_K608003) following the rules of the BioBrick RFC 10 assembly protocol. Positive transformants were selected on growth media with chloramphenicol and ampicillin and further verified by colony PCR with primers VF2 and VR.

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/2.jpg)

**NDM MBL (doi.org/10.3390/ijms232416083)**

**5) Test and Evaluate**

To test the newly generated constructs, we applied the following methods

**• Antibiograms** – antibiograms were prepared following the EUCAST criteria, utilizing discs loaded with imipenem, meropenem, and imipenem + EDTA. The distinction in zone diameters between imipenem and imipenem + EDTA indicated a MBL production in our transformants.

**• Meropenem degradation by cell free extract** – we prepared cell free extracts from IMP and NDM producing strains using a combination of centrifugation (2700 g for 10 min) and filtration (0.22 um filter). The complete elimination of cells was confirmed by inoculating portions of the cell-free extract onto agar plates with a suitable growth medium. Subsequently, we examined the extract's ability to degrade carbapenems by treating MH medium with 50 µg/ml of meropenem. The residual antibiotic levels were determined using the agar well dilution method, and notably, complete meropenem degradation was observed.

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/3.png)

**Agar well dilution method**

**• Carbapenem hydrolysis monitoring in real time** – we applied ultraviolet-visible (UV-Vis) spectroscopy to monitor the degradation of meropenem. A sample containing the cell-free extract and meropenem was prepared in a quartz cuvette, and we measured the absorbance at 298 nm for 10 minutes using a JENWAY 6715 UV-Vis spectrophotometer. The slope of the resulting line was used as an indicator for the enzymatic activity of our MBL extract.

**6) Iterate, Refine and Improve**

Based on the findings from our initial tests, we concluded that both of the newly constructed variants resulted in the production of functional MBL enzymes. Notably, the hybrid NDM-2&5 variant exhibited the most significant MBL activity, as indicated by the antibiograms and kinetic curves. To assess its performance relative to the original VIM-2 utilized in 2022, we conducted a broth dilution assay to determine the effective MIC (Minimum Inhibitory Concentration) of meropenem for strains producing VIM-2 and NDM-2&5. The results revealed that E. coli DH10B *blaVIM-2* exhibited no growth inhibition at 25 µg/ml of meropenem, while the *E. coli* DH10B *blaNDM-2&5* clone displayed inhibition at concentrations exceeding 200 µg/ml, representing a **substantial improvement of more than eightfold.**

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/4.png)

**Broth dilution assay to evaluate the meropenem’s MIC of our MBL producers**

However, we believed that we had not fully harnessed the potential of our chosen MBLs. To maximize their capabilities, our first step was to employ a directed evolution strategy to enhance our NDM-2&5 enzyme. We opted for a **dual gene-specific mutator system capable of introducing transition mutations at consistent frequencies within a target sequence *in vivo.*** (Seo D, Koh B, Eom GE, Kim HW, Kim S. A dual gene-specific mutator system installs all transition mutations at similar frequencies *in vivo*. Nucleic Acids Res. 2023 Jun 9;51(10):e59. doi: 10.1093/nar/gkad266. PMID: 37070179; PMCID: PMC10250238.). To utilize its mutagenic potential, first we cloned our *blaNDM-2&5* construct in a plasmid with T7 promotor and RBS (BBa\_K525998). While this approach would have effectively introduced random mutations into our sequence, we also needed a straightforward method for screening our clones to identify MBL production. Our attempts to induce expression using the T7 promoter in our construct within *E. coli* KRX cells did not yield MBL production after L-rhamnose induction, as indicated by test antibiograms. To address this challenge, we opted to subclone our fragment into a pSB1A2 vector (BBa\_J23101) that includes the J23101 Anderson constitutive promoter. We anticipated that this promoter would facilitate protein expression, while the T7 promoter could be reserved for mutagenesis purposes. To test this hypothesis, we created a construct comprising J23101-T7-RBS followed by the coding sequence of amilGFP (BBa\_K592010). Upon observation of the transformants under blue light, clear signs of amilGFP expression became evident.

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/5.png)

**Construct J23101-T7-RBS-amilGFP CDS in pSB1A2 (E. coli DH10B cells, no gene for T7 RNA polymerase)**

Once we validated the functionality of our approach, we proceeded to subclone the T7-RBS-*blaNDM-2&5* fragment into the BBa\_J23101 plasmid. Positive transformants were chosen based on their meropenem resistance and confirmed through colony PCR verification. In this configuration, MBL production occurred consistently from the J23101 promoter, facilitating straightforward functional assessments.

Regrettably, the constructs based on pSB1A2 proved unsuitable for mutagenesis. Firstly, this vector is characterized by a high copy number, whereas the system necessitated a low copy number target. Additionally, it contains a standard β-lactamase gene, conferring resistance to ampicillin, which could have interfered with our MBL when assessing our extract for ampicillin degradation. To address these challenges, we carried out subcloning of our T7-RBS-amilGFP and T7-RBS-*blaNDM-2&5* fragments into the low copy number SC101 ori-based vector pSB4K5 (kanamycin resistance).

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/6.png)

**Construct J23101-T7-RBS-amilGFP CDS in pSB4K5 (*E. coli* DH10B cells, no gene for T7 RNA polymerase)**

The resulting constructs were employed for mutagenesis purposes. In this process, plasmid DNA from our test construct was extracted from positive transformants and subsequently reintroduced into competent cells that harbored plasmids pDae029 (eMutaT7[TadA-8e] – Addgene 187620), pDae069 (eMutaT7[PmCDA1] with an optimized RBS – Addgene 187621), or pDae079 (eMutaT7[transition] – Addgene 187622). Following this, amilGFP-expressing clones were cultivated on media containing both chloramphenicol and kanamycin, and mutagenesis was initiated by the addition of L-arabinose. This procedure underwent three rounds of mutagenesis, with colony monitoring conducted after each round. Multiple clones with altered colour, brightness, and/or size were readily detectable after the second round. Their number was highest in the cells containing the pDae079 mutagenesis plasmid.

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/7.jpg)

**Initial transformants and the resulting colonies with altered colour, brightness, and/or size due to the random mutagenesis**

Next, we re-transformed the pSB4K5-J23101-T7-RBS-*blaNDM-2&5* construct into competent *E. coli* DH10B cells with pDae079. Three positive transformants were selected on media with ampicillin and 10 rounds of mutagenesis were performed with each of them. Aliquots of each clone from every round were combined and incubated in liquid LB medium enriched with elevated meropenem concentrations. Remarkably, we witnessed bacterial growth in cultures exposed to 600 µg/ml of meropenem, representing **a remarkable improvement of more than 24-fold compared to the original VIM-2 construct.**

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/8.png)

**The improved activity of the mutagenized NDM-2&5 MBL**

To validate the effectiveness of the newly developed CADABRA extract based on NDM-2&5 in eliminating carbapenems from hospital wastewater, we replicated last year's experiment using artificial urine contaminated with 750 µg/ml of meropenem. We followed the same procedure, and this time **we observed the complete removal of antibiotics from the sample. Our first goal was accomplished!**

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/9.png)

**NDM-CADABRA vs artificial urine with meropenem**

Concerning the challenge of ampicillin degradation, we evaluated the performance of the original VIM-2 CADABRA and the newly prepared, non-mutagenized NDM-2&5 CADABRA against elevated ampicillin concentrations (1 mg/ml) within a short treatment duration (45 minutes). **The outcomes demonstrated that our new version surpassed the original by at least fourfold.**

![](https://static.igem.wiki/teams/4855/wiki/notebook/engineering/10.jpg)

**NDM-2&5- and VIM-2-based CADABRA against high levels of ampicillin**

With this we demonstrated our engineering success.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4855/wiki/protocols/1.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/2.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/3.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/4.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/5.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/6.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/7.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/8.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/9.png)

![](https://static.igem.wiki/teams/4855/wiki/protocols/15.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

*In our search for ways to improve the CADABRA enzyme cocktail, we obtained the following results*

**1) Designing, synthesizing, and cloning two new MBL coding sequences**

We designed two new metallo-β-lactamase (MBL) coding sequences. The first one featured the E. coli codon-optimized version of the IMP-1 MBL (Q79MP6). The second construct represented an artificial fusion of the NDM-2 (F2YZ26) and NDM-5 (A0A2S1PIF3) variants, known to exhibit the highest meropenem hydrolyzing capabilities based on existing literature. In both coding sequences, C-terminal 6xHis tags were incorporated for subsequent purification purposes. The two sequences were synthesized as gBlock fragments by IDT.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/1.png)

**Schematic overview of both gBlock fragments**

Following the acquisition of the fragments, they were subjected to dilution and subsequently underwent PCR amplification using VF2 and VR primers. In this process, Phusion polymerase was utilized with a limited number of cycles to minimize the likelihood of occurrence of unintended mutations.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/2.png)

**PCR-amplified MBL fragments**

Subsequently, both PCR products underwent purification with the Zymo DNA Clean & Concentrator kit, followed by elution in 20 µl of nuclease-free H2O. These products were then digested using XbaI and PstI, followed by purification as previously described. Concurrently, plasmid DNA was obtained from part BBa\_K608003, which contains a strong promoter and a medium RBS. This DNA was treated with SpeI and PstI, and afterward purified from the restriction endonucleases. The quantities of all digested DNA molecules were determined using Quantus (Promega), and ligations were conducted using a 3:1 insert-to-vector molar ratio. Positive transformants were chosen on dishes with chloramphenicol and ampicillin, as only the MBL-positive clones would be capable of eliminating the latter antibiotic.

**2) Confirming that both new constructs express MBLs**

To confirm that both new constructs produce functional MBLs, we prepared antibiograms according to the EUCAST guidelines, utilizing discs with imipenem, meropenem, and imipenem + EDTA. The distinction in zone diameters between imipenem and imipenem + EDTA (>> 8 mm) indicated a MBL production in our transformants.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/3.png)

**Illustrative antibiogram for phenotypic MBL detection**

**3) Assessing the meropenem degradation capabilities of IMP-1 and NDM-2&5 cell free extracts**

All cultures were established using *E. coli* DH10B cells containing the respective IMP-1 and NDM-2&5 expression constructs. Cultivation was conducted in LB medium supplemented with meropenem. Following inoculation, the cultures were incubated for a minimum of 16 hours at 37°C and 250 rpm on an orbital shaker. Post-cultivation, centrifugation was employed at 2700 g for 10 minutes, followed by filtration through a 0.22 µm filter to yield a cell-free supernatant.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/4.png)

**Cell supernatant filtration through 0.22 µm filter**

Following this step, we prepared Muller-Hinton broth, enriched with 50 µg/ml of meropenem, and subjected it to treatment with CADABRA. To assess the extent of antibiotic degradation achieved by our MBL enzyme, we created petri dishes filled with 0.7% LB agar medium and inoculated them with *E. coli* DH10B containing pSB1C3-amilGFP as an indicator strain. Once the agar had solidified, we created small wells in it and introduced serially 2-fold diluted portions from the treated sample into these wells. Before this procedure, we blocked MBL activity by adding EDTA.

All cultures were established using *E. coli* DH10B cells containing the respective IMP-1 and NDM-2&5 expression constructs. Cultivation was conducted in LB medium supplemented with meropenem. Following inoculation, the cultures were incubated for a minimum of 16 hours at 37°C and 250 rpm on an orbital shaker. Post-cultivation, centrifugation was employed at 2700 g for 10 minutes, followed by filtration through a 0.22 µm filter to yield a cell-free supernatant.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/5.png)

**Preparation of Petri dishes for the agar well dilution method**

Subsequently, the petri dishes were gently placed in an incubator and allowed to incubate overnight at 37°C. The following day, all the dishes were examined under blue light transillumination to check for the presence of growth inhibition zones. None were detected, indicating complete hydrolysis of the antibiotic by the corresponding MBLs.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/6.png)

**Meropenem degradation by cell free extracts**

**4) Monitoring the meropenem degradation in real time**

To monitor the meropenem degradation process, we utilized ultraviolet-visible (UV-Vis) spectroscopy. For this purpose, we first procured quartz cuvettes with appropriate volumes.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/7.png)

**Quartz cuvettes**

Next, we optimized the conditions for meropenem detection, looking for the corresponding peak at 298 nm

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/8.jpg)

**Monitoring of the meropenem concentrations in water samples via UV-Vis spectroscopy**

We created a sample by combining the cell-free extract with meropenem inside a quartz cuvette and recorded absorbance measurements at 298 nm for a duration of 10 minutes using a JENWAY 6715 UV-Vis spectrophotometer. The slope of the resulting line was employed as an indicator of our MBL extract's enzymatic activity.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/9.png)

**Illustrative curve of meropenem’s degradation monitored via UV-Vis spectroscopy**

Results indicated that NDM-2&5 outperforms VIM-2 and IMP-1.

**5) Assessing the MICs of meropenem for VIM-2 and NDM-2&5 producers.**

A broth dilution assay was used to determine both MICs.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/10.png)

**Broth dilution assay to find the MIC of meropenem for MBL producers**

**6) Optimization of the NDM-2&5 MBL via directed evolution**

To further elevate the performance of the new CADABRA based on NDM MBL, we opted to employ directed evolution for the purpose of enhancing the enzyme's activity. To execute this, we cloned both the coding sequences of NDM-2&5 and IMP-1 into a plasmid featuring a T7 promoter and RBS (BBa\_K525998) while adhering to the BioBrick RFC 10 assembly guidelines.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/11.png)

**Schematic representation of the T7 constructs**

All cloning procedures were carried out in *E. coli* KRX cells, which harbor the T7 RNA polymerase gene controlled by an L-rhamnose-inducible promoter. In this instance, identifying positive transformants through incubation on Petri dishes with β-lactam antibiotics was unfeasible, prompting us to resort to colony PCR for verification.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/12.png)

**Identification of positive clones via colony PCR**

Subsequently, we attempted to assess the functional performance of our constructs by generating antibiograms. In this instance, we cultivated the strains in MH liquid medium supplemented with L-rhamnose to induce the T7 promotor. The remaining part of the procedure was performed as usual. Regrettably, we did not observe any indications of MBL production in neither of our PCR-positive transformants.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/13.png)

**All antibiograms showed no MBL activity**

To address this challenge, we made the decision to design novel constructs in which we placed a J23101 constitutive Anderson promoter upstream of the T7-RBS-*blaNDM2&5* fragment. Our expectation was that this promoter would promote protein expression, allowing the T7 promoter to be dedicated to mutagenesis. To evaluate this concept, we constructed a hybrid promotor comprising J23101-T7-RBS, followed by the coding sequence of amilGFP (BBa\_K592010).

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/14.png)

**Schematic representation of the j23101-T7 constructs**

Upon examination of the transformed cells under blue light, conspicuous evidence of amilGFP expression became apparent.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/15.png)

**The hybrid J23101-T7 promotor is functional as indicated by the amilGFP positive clones**

After we confirmed the effectiveness of our approach, the next step involved subcloning the T7-RBS-*blaNDM2&5* 5 fragment into the BBa\_J23101 plasmid. Subsequently, both of our constructs were introduced into the low copy number SC101 ori-based vector, pSB4K5 (kanamycin-resistant), resulting in the final constructs required for *in vivo* mutagenesis.

The *in vivo* mutagenesis was performed using a dual gene-specific mutator system capable of introducing transition mutations at consistent frequencies within a target sequence *in vivo* that was available for ordering from Addgene. (Seo D, Koh B, Eom GE, Kim HW, Kim S. A dual gene-specific mutator system installs all transition mutations at similar frequencies *in vivo*. Nucleic Acids Res. 2023 Jun 9;51(10):e59. doi: 10.1093/nar/gkad266. PMID: 37070179; PMCID: PMC10250238.).

We performed 3 rounds of mutagenesis for the J23101-T7-RBS-amilGFP construct and ten rounds for the J23101- T7-RBS-*blaNDM2&5* sequence using DH10B cells containing pDae079.

![](https://static.igem.wiki/teams/4855/wiki/notebook/results/16.jpg)

**Monitoring the mutagenesis efficiency at the different rounds via the number of colonies with altered colour, brightness, and/or size**

We combined samples from the NDM-2&5 constructs taken from each mutagenesis round and incubated them in liquid LB medium with increased meropenem concentrations. In contrast to the unmodified version, which could only grow on medium with up to 200 µg/ml, we observed bacterial growth in cultures exposed to 600 µg/ml of meropenem after the mutagenesis.

{% endblock %}