iGEM 2021 — Team Bulgaria

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# Team:Bulgaria/Notebook

# Notebook

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# Team:Bulgaria/Engineering

# Engineering

Improvise Adapt Overcome.

### Working cells no more

One of the very first things we learned to do is make chemically competent cells using DH10B Escherichia coli cells, our first few tries were successful but very quickly things took a wrong turn. We noticed that our cells were taking longer and longer to reach the right absorbance at 600nm. We decided to check if the problem was with the LB powder we used to make the medium, but the results were the same. We then tried using a different strain, KRX E. coli cells. The cells grew normally and from then on, our experiments were done using this strain.

### Say goodbye to the constructs

As we mentioned, our cells were dying before we could make them competent. We didn't notice that though until after the first experiment where we lost the constructs we were working with. In order to fix this problem, we tried amplifying the minimal amount that was left, through PCR, after which we amplified them using a standard vector. Our attempt was successful, and we managed to restore and use the lost constructs.

### Antibiogram swabs

During our experiments we wanted to conduct an antibiogram. But, because of the Covid-19 pandemic, there was an influx of swab usage which led to our distributors being sold out. Still, we needed the swabs in order to conduct our experiment and we came up with the idea of autoclaving normal q-tips to use instead of the swabs. The only downside to this was that the q-tips are short, but we used a sterile tweezers, when needed.

### Electronics raise the temperature, **go figure!**

While using our new device that can measure bacterial growth kinetics on solid media, we noticed that the temperature in our incubator increased. Obviously, we came to the conclusion that all the extra electronic parts, more specifically the motor, working inside the incubator contributed to the extra heat. To combat this, we opened the air valve 40% which decreased the temperature, back to the desired 37°C.

#### Ask

We asked ourselves which are the most likely gene candidates for antibiotic resistance to be found in wastewaters, due to the Covid-19 pandemic and the over-prescription of various antimicrobial drugs.

#### Research

After screening multiple publications on the topic, we organized a consultation meeting with the leading expert in clinical microbiology in Bulgaria (Prof. Tanya Strateva, MD), where we discussed our observations. Summarizing the literature data, and the opinion of the expert, we decided to concentrate on genetic determinants for resistance to various types of b-lactams (the focus being on the class of metallo-beta-lactamases and other carbapenem-inactivating enzymes) and quinolones (focus on all mechanisms responsible for resistance to the third-generation fluoroquinolone, levofloxacin, because of it’s wide usage in most Covid treatment protocols).

#### Imagine

Quickly, we realized that two major types of resistance mechanisms exist in nature. One of them relies on presence of specific genetic determinants that are responsible for drug resistance and presence of chromosomal mutations that alter the sequences of normally found, in the given microorganism genome, genes and, thus, resulting in antibiotic resistance. Studying the selected major types of resistance, we realized, that the beta-lactam resistance is mainly mediated by the presence of various types of beta-lactamases. On the other hand, the most prominent mechanism for quinolone resistance is based on mutations in GyrA (DNA gyrase) and ParC (topoisomerase IV) genes. Searching for appropriate solution to monitor these two types of resistance mechanisms, we realized that several solutions, compatible with our equipment and expertise limitations, exist:

• Shotgun metagenomic sequencing;

• PCR-based screenings;

• Genetic library construction and selection of Escherichia coli clones on plates with antibiotics

#### Plan

After intensive discussions, we decided to use a three-step approach. First, we isolated total DNA from wastewater samples, and we sent two samples for NGS sequencing, using the DNB-seq technology that is commercialized by the BGI Genomics Company. This is the most direct approach, but its main limitations lie in the relatively high-cost per sample, and the long turnover time. So far, we haven’t yet received our results. In the meantime, we prepared a bioinformatics pipeline that allows the detection of antimicrobial resistance genes. Furthermore, it is worth to be mentioned, that the antibiotic resistant genes represent only a small percentage of the bacterial genomes in our sample, thus, leading to the fact that the vast majority of our sequencing reads do not provide useful information. To overcome this difficulty, we developed the second PCR-based approach. To specifically amplify genetic resistance determinants, we focused on the specific genetic structures, called Class I integrons, since they carry cassettes with antimicrobic resistance genes that can be transmitted from one strain to another. Using conservative elements in the structure of Class I integrons, we developed a set of PCR primers that can amplify the resistance gene cassettes. After the amplification of all cassettes in our sample of interest, we planned to clone them in E. coli and then to select clones, on petri dishes with various antibiotics, like meropenem and levofloxacin (the meropenem was selected, because this was the only carbapenem that we managed to get our hands on). Using this strategy, we can quickly, and at almost zero cost, identify specific types of resistant determinants in the integron gene cassettes. A major drawback is the fact that, using this approach, we will miss every antibiotic resistance gene, that is not located inside integrons. Nevertheless, this is not really a disadvantage, because many bacterial species naturally contain resistant determinants, but are non-pathogenic. Focusing on integrons allows us to obtain information about resistant determinants, that can be easily transferred to bacterial pathogens of clinical importance. Due to this, we selected this second approach as our major technique for this project.

As a third option, for monitoring the levofloxacin resistance levels, we decided to create a specialized cloning-based detection tool, that relies on amplification and cloning of the QRDR regions into a reporter vector. Last, but not least, we ordered a specific primer pair for vancomycin and colistin, since the other approaches are not well-suited to explore the resistance levels against these drugs.

#### Create

We concentrated our work on the creation of two prototypes:

1. Cloning device for the Class I integron resistance gene cassettes.

We designed a set of the following seven primers:

| Name | Sequence |
| --- | --- |
| Int\_class1\_1F | ACACCGTGGAAACGGATGAA |
| Int\_class1\_2F | GATGAAGGCACGAACCCAGT |
| Int\_class1\_3F | GGAAACGGATGAAGGCACGA |
| T7\_Int\_class1\_2F | TAATACGACTCACTATAGGATGAAGGCACGAACCCAGT |
| Int\_class1\_1R | TCTAAGAGCGGCGCAATACG |
| Int\_class1\_2R | CGGTTGGAAGCTGTCGATTG |
| Int\_class1\_3R | CCTCGAAGAACCGCACAATC |

That allows the desired amplification. All forward primers, located inside the highly conservative Integrase I gene, and all reverse oligos lie inside the sulI gene that are conservative parts of the integron Class I structure. For the primer design, we used the Primer BLAST tool, feeding it with a large number of intI and sulI sequences, in order to ensure that our oligonucleotides won’t be affected by any common sequence variations. One of the primers, T7\_Int\_class1\_2F, also contains a T7 promotor. The design was selected to allow us to increase the expression levels, using a specific strain of E. coli, that contains T7 polymerase (like the KRX cells from Promega).

2. For the QRDR mutations, we developed a specific vector, that contains the gyrA gene. We selected this gene, because a resent publication, entitled “Pandemic fluoroquinolone resistant Escherichia coli clone ST1193 emerged via simultaneous homologous recombinations in 11 gene loci” (doi: 10.1073/pnas.1903002116), indicated that the vast majority of quinolone resistant E. coli isolates carry two specific mutations in this gene (S83 and D87). Our design allows the amplification of the QRDR region of E. coli genomes in wastewaters, and then cloning of these PCR products in a reporter vector that contains the remaining part of the gyrA gene. After transformation in E. coli competent cells, we expect that the number of quinolone-resistant clones will correspond with the overall quinolone resistance levels.

3. We obtained genomic DNA from a carbapenem-resistant clinical isolate that belongs to the Klebsiella pneumoniae we sent for whole genome sequencing to test if we can identify the cause of its carbapenem resistance.

#### Test

To test our prototypes, we performed the following experiments:

1. We received genomic DNA from a recently sequenced clinical isolate - Pseudomonas aeruginosa, that was found to harbor Class I integron that contains VIM-2 metallo-beta-lactamase (“Characterization of a Bulgarian VIM-2 metallo-β-lactamase-producing Pseudomonas aeruginosa clinical isolate belonging to the high-risk sequence type 111” doi.org/10.1080/23744235.2021.1934531). Using our primers, we succeeded in amplifying the VIM-2-containing cassette and cloning it into a specially designed T-cloning vector. We focused on the TA cloning approach, since, in principle, we do not know what type of cassettes we are going to find in our samples. Because of this, we cannot use the standard set of restriction endonucleases, supported by the BioBrick standard, since it is likely that some of them will cut inside the cassettes of interest. The TA cloning eliminates this possibility and it’s also cheap and fast. Transferring this cassette into E. coli, we succeed to also carry over the resistance to meropenem.

2. We successfully generated the, required for this approach, vector and designed three sets of primers that amplify the QRDR region of the gyrA gene in many sequenced E. coli genomes:

| Name | Sequence |
| --- | --- |
| Primer F1 | GAGCGACCTTGCGAGAGAA |
| Primer F2 | ATGAGCGACCTTGCGAGAG |
| Primer F3 | AGCGACCTTGCGAGAGAAAT |
| Primer R1 | CGTTAATGATTGCCGCCGTC |
| Primer R2 | GCTTCTTCAATACCGCGACG |
| Primer R3 | GACCGTTAATGATTGCCGCC |

We were lucky enough to find that Primer R2 has an almost completed HintIII restriction site in its 5’ end. We decided to use this site in our cloning approach. To the 5’ end of the forward primer, we added a restriction site for XbaI. In order to clone the QRDR region, the PCR products were digested with these two enzymes and then ligated into the vector, that contains the remaining part of the gyrA gene. Unfortunately, we were not able to fully evaluate this prototype, because the wild-type gyrA gene, located on the chromosome of E. coli, shows a dominant phenotype of quinolone susceptibility, and, because of this, no resistant clones were observed.

3. We received 1 Gb sequencing information that consisted of 2x150 bp paired-end reads from BGI genomics. We used it to assemble the draft genome sequence of this isolate. Next, we used a specialized program for detection of antibiotic resistance genes in assembled contigs to check the overall resistome of this strain. We identified blaKPC-2 gene that encodes for carbapenem-hydrolyzing class A beta-lactamase KPC-2 enzyme that explains the carbapenem resistance phenotype.

That allows the desired amplification. All forward primers, located inside the highly conservative Integrase I gene, and all reverse oligos lie inside the sulI gene that are conservative parts of the integron Class I structure. For the primer design, we used the Primer BLAST tool, feeding it with a large number of intI and sulI sequences, in order to ensure that our oligonucleotides won’t be affected by any common sequence variations. One of the primers, T7\_Int\_class1\_2F, also contains a T7 promotor. The design was selected to allow us to increase the expression levels, using a specific strain of E. coli, that contains T7 polymerase (like the KRX cells from Promega).

#### Improve

We applied the following improvements:

1. We modified the TA cloning vector in a way that it utilizes the mRFP1 coding sequence as a color-based selection tool. That substitutes for the initial LacZα fragment that we amplified pUC19 vector (you can see our Parts list for details about the two vectors). This improvement allowed us to eliminate the addition of IPTG and X-Gal for screening purposes.

2. In order to use the reporter vector we created, we needed a strain of E. coli that could suppress its chromosomal gyrA gene copy. Since this gene is essential, we selected a specific strain, described in literature under the name of KNK453. These cells contain a special thermo-sensitive mutation in the gyrA gene, that allows the protein to function at 30C, but turns it inactive at temperatures over 37C. Unfortunately, we were not able to obtain this strain in time, due to logistic difficulties. So, this part of our experimental design remains untested at the current moment.

3. Proving that our sequencing-based approach works, we sent two waste water samples for shotgun metagenome sequencing.

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# Team:Bulgaria/Experiments

Bootstrap 5 generator example page

# Protocols

![image](https://static.igem.org/mediawiki/2021/4/4b/T--Bulgaria--lab.jpeg)

![image](https://static.igem.org/mediawiki/2021/1/1c/T--Bulgaria--work--lab.jpeg)

All the experiments performed are described in our Engineering section. Here you can find all protocols we used in our work.

# Miniprep Plasmid Purification

Notes:

iGEM Bulgaria used the GeneJET Plasmid Miniprep Kit from Thermo Scientific for all plasmid isolations.

Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.

Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.

Centrifuge for 5 min to pellet cell debris and chromosomal DNA.

Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.

Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.

Repeat the wash procedure using 500 uL of the Wash Solution.

Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.

Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.

Discard the column and store the purified plasmid DNA at -20°C.

# PureLink® Quick Gel Extraction and PCR Purification Combo Kit

Notes:

iGEM Bulgaria used the PureLink® Quick Gel Extraction and PCR Purification Combo Kit from Thermo Scientifir for all reaction clean-ups, PCR clean-ups and agarose gel extraction procedures.

Purifying PCR fragments or reaction clean-up

Combine. Add 4 volumes of Binding Buffer (B2) with isopropanol to 1 volume of a PCR sample (50–100 uL). Mix well.

Load. Pipet the sample into a PureLink® Clean-up Spin Column in a Wash Tube. Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through.

Wash. Re-insert the column into the Wash Tube and add 650 uL Wash Buffer (W1) with ethanol. Centrifuge the column at >10,000 × g for 1 minute.

Remove ethanol. Discard the flow-through and place the column in the same Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes.

Elute. Place the column into a clean 1.5-mL Elution Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the column at room temperature for 1 minute. Centrifuge the column at maximum speed for 1 minute.

Store. The elution tube contains the purified PCR product. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.

Purifying DNA from Gels Using a Centrifuge

Excise. Use a clean, sharp razor blade to excise a minimal area of gel containing the DNA fragment of interest.

Weigh. Using a scale sensitive to 0.001 g, weigh the gel slice containing the DNA fragment.

Solubilize. Add Gel Solubilization Buffer (L3) to the excised gel in a tube as indicated in the table. Incubate the tube at 50°C for 10 minutes (or longer for large gel slices and high concentration gels), and invert the tube every 3 minutes. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes. Add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.

Load. Pipet the dissolved gel piece onto a column inside a Wash Tube. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Note: The column reservoir capacity is 850 uL. Use 1 column per 400 mg of agarose gel.

Wash. Add 500 uL Wash Buffer (W1) containing ethanol to the column. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes. Discard the flow-through.

Elute. Place the column into a Recovery Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the tube for 1 minute at room temperature. Centrifuge the tube at >12,000 × g for 1 minute.

Store. The elution tube contains the purified DNA. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.

# Ligation

Add the following reagents to a PCR tube: Reagent Amount 5X Ligase Reaction Buffer Vector DNA 50-100 ng Insert DNA (1:1 or 3:1 ratio) ExpressLink™ T4 DNA Ligase 5 units (in 1 uL) autoclaved distilled water to 20 uL

Mix gently. Centrifuge briefly to bring the contents to the bottom of the tube.

Incubate at room temperature for 5 - 15 minutes.

# Glycerol Stocks

Pick a single colony of the clone off of a plate and grow an overnight in the appropriate selectable liquid medium (e.g., LB amp).

Make a label (clone ID # and date) for the construct.

Add 0.5ml of the o/n culture to 0.5ml of 80% sterile glycerol in the sterile screw cap microcentrifuge tube.

Screw a lid onto the tube and write the clone ID # on the lid of the tube.

Vortex.

Freeze the glycerol stock at –80°C.

To streak out from a glycerol stock:

Determine the location (-80°C tower #, box #, row #) of the construct.

Take the tube to the place that you intend to streak the clone out

Flame a metal inoculating loop until it is red hot.

Scrape off a portion from the top of the frozen glycerol stock and streak it onto your plate.

Return the construct to the –80°C.

# gBlock Fragment Amplification

Phusion® DNA Polymerase amplification reaction

Nuclease-free

H2O Adjust to final 50 uL

5X Phusion HF

10 uL 10 mM dNTPs

1 uL 10 uM Forward Primer

2.5 uL 10 uM Reverse Primer

2.5 uL gBlocks® Gene Fragments

0.1–1.0 ng Phusion® DNA Polymerase 0.5 uL

Total volume 50 uL

PCR program:

Initial denaturation: 98°C, 30 seconds

Cycle start:

Denaturation 98°C, 10 seconds

Annealing 60°C, 30 seconds

Extension 72°C, 20 seconds per kb

Cycle end (25-35 cycles)

Final extension 72°C, 5 minutes

Hold 4°C

# TSS Competent Cells Preparation and Transformation

This protocol of competent cell preparation follows the TSS preparation described by Chung & Miller (Chung, C. T. & Miller, R. H.; Preparation and storage of competent Escherichia coli cells Methods Enzymol, 1993, pp. 621-627). We have tested the method described below with two different E. coli strains: KRX. Nevertheless, all optimizations have been made using the KRX strain.

Materials:

- dimethyl sulfoxide (DMSO)

- polyethylene glycol 6000 (PEG 6000)1

- 1M MgCl2 stock solution

- 1M MgSO4 stock solution

- LB medium (liquid and 1,5% agar)

- SOC liquid medium2

- antibiotic stocks

Equipment:

- Orbital Shaker at 37°C

- Centrifuge (equipped with rotor for 50 ml Falcon tubes with up to 2700 g support)

- Spectrophotometer

- Thermoblock

Consumables:

- 50 ml Falcon Conical Centrifuge Tubes

- 50 ml sterile Erlenmeyer flask3

- 500 ml sterile Erlenmeyer flask

Protocol

Competent cells preparation

Day 1:

1) Inoculate 5 ml of liquid LB medium with E. coli DH5alfa in a 50 mL Erlenmeyer flask and culture overnight at 37°C with shaking at 250 rpm.

2) Prepare 1x TSS buffer (if you do not have one at -20°C)

To make 20 ml:

• 2 g PEG 60001

• 0.3 ml 1M MgCl2

• 0.3 ml 1M MgSO4

• add LB media to 19 ml and mix until PEG is dissolved4

• Filter sterilize (0.22 um filter)

• add 1 ml DMSO (it is sterile by itself)

Store at at -20°C (should be good for a few months)

Day 2:

1) Prepare 50 ml of liquid LB medium in a sterile 500 ml Erlenmeyer flask.5

2) Seed 0.25 ml from the overnight culture into the 500 ml flask and incubate at 37°C with 250 rpm shaking.6
At this point you should take out the 1x TSS buffer, melt it and then store it on ice until it is needed. Additionally, you have to cool down your centrifuge to 2-4°C and put one 50 ml Falcon tube on ice.

3) Cultivate until the optical density of the culture at 600 nm wavelength is 0.3 to 0.4 (0.3 - OD600 - 0.4)7
TSS buffer should be chilled by this point.

4) Once the proper optical density has been achieved, transfer 50 ml of the culture into one 50 ml Falcon Conical Centrifuge Tube and centrifuge it under 2,700xg for 10 min at 2-4°C.

5) Resuspend the cell pellet in 1 ml of pre-chilled 1x TSS buffer with gentle pipetting (use blue tip). Distribute 100 ul of TSS suspended cells to labeled and pre-chilled 1.5 ml Eppendorf tubes while ensuring the cells remain well mixed.
Cells can be used immediately, or stored at -80°C.

Competent cells transformation

1) Thaw TSS cells on ice (if they are not used immediately after preparation) . Thaw some SOC liquid medium.8

2) Add DNA, pipette gently to mix (if you are adding small volumes like ~ 1 ul, be careful to mix the culture well). Do not use more than 10 ul sample (1/10 of the cell volume).

3) Incubate for 30 minutes on ice.

4) Heat shock the cells for 45 seconds at 42°C.9

Note: According to the original TSS paper, this step is optional and may actually reduce transformation efficiency. Nevertheless, in our optimization experiments the term shock step increased the observed transformation efficiency for DH5alfa ~ 100 fold (measured when transformed with 300 pg pSB1C3).

5) Incubate cells on ice for 2 min.

6) Add 0.9 ml SOC medium at room temperature.2

7) Incubate for 1 hour at 37°C on shaker.10

8) Spread 50 ul onto a pre warmed (37°C) LB plate with appropriate antibiotic.11

9) Grow overnight at 37 °C.12

Notes:

1. Different types of PEG can be used. Actually, the original paper suggest PEG 3350 as the most efficient option. On the other hand most of the modern variations of this protocol use PEG 8000. We have tested PEG 6000 since it was the only one we had in stock.

2. We have also used LB medium + 10 mM MgCl2 +10 mM MgSO4 + 20mM glucose instead SOC.

3. For optimal aeration use cultivation vessels with a 10 times (down to a minimum 5 times) higher volume than your culture.

4. The original paper suggest that you should adjust the pH to 6.5. We have never done that.

5. For 10 aliquots of cells, 100 ul each. Scale up or down according your needs.

6. If you want to speed up a bit you can use 1/100 volume for inoculation -500 ul (but not more).

7. We always try to take the cells at OD ~ 0.4. The incubation takes app. 3 h

8. SOC can be contaminated quite easily so store it in small aliquots at -20°C.

9. The optimal time of the heat shock depends on many things - strain, different types of tubes, thermo block or water bath at 42°C and so on. If you use different experimental setup, optimize it - try 30 s, 45 s, 60 s, 90 s, 120 s and 60 min on ice without T shock.

10. Can be shorten to 45 min

11. Save the rest of the transformants in liquid culture at 4°C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.

12. Or at lower temperature if your construct contains some T sensitive elements.

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# Team:Bulgaria/Results

# Results:

**1.** Introduction of novel ingredients in the cultivation media for improved monitoring of the bacterial growth on solid media.
We tested two different compounds (titanium dioxide and activated charcoal) that lead to white and black agar respectively. Next, we assembled different expression constructs, with chromoproteins, to see if they show improved visibility compared to normal LB medium. You can see some representative results, on the following pictures.

![image]( https://static.igem.org/mediawiki/2021/2/26/T--Bulgaria--GFPBlack.jpg)

![image]( https://static.igem.org/mediawiki/2021/f/fa/T--Bulgaria--RFPWhite.png)

The overall conclusion is that red chromoproteins are best observed on white media, while the yellow ones show optimal visibility on the black agar.

**2.** Building of a novel system for continuous kinetic measurement of cell growth on a petri dish.
We measured the difference in growth kinetics between E. coli cells on the novel agars we mention above and bacterial growth on normal LB agar. We also noted the contrast difference of the different types of genetically modified cells with different colored light sources.

![image](https://static.igem.org/mediawiki/2021/a/a5/T--Bulgaria--HardwareBlue.png)

![image]( https://static.igem.org/mediawiki/2021/5/51/T--Bulgaria--HardwareGreen.png)

![image]( https://static.igem.org/mediawiki/2021/9/9d/T--Bulgaria--HardwareAll.png)

Images from the camera

**3.**Sequencing the genome of a clinical carbapenem resistant strain Klebsiella pneumoniae.

We received 1 Gb sequencing information that consisted of 2x150 bp paired end reads from BGI genomics. The resulting raw read pairs were
uploaded to the [Galaxy online platform](https://usegalaxy.eu/). The read pairs were inspected with FastQC and then trimmed for adapter sequences and poor-quality bases by Trimmomatic. Initial de novo assembly of the processed reads was carried out using the SPAdes assembler with default settings. SPAdes generated a 5.52 Mb assembly comprising of 82 contigs larger than 1000 bp with an N50 value of 198,180 bp and an average GC content of 57.2% (evaluated by QUAST). Next we investigated the resistome of this strain, using the tool ABRicate. All localized antibiotic resistance determinants are shown in the following table:

![image](  https://static.igem.org/mediawiki/2021/e/e9/T--Bulgaria--Table.jpg)

We successfully identified the reason for carbapenem resistance, the blaKPC-2 gene that encodes for carbapenem-hydrolyzing class A beta-lactamase KPC-2 enzyme.

**4.** We designed primers that can amplify the variable gene cassettes of Class I integrons:

| Name | Sequence |
| --- | --- |
| Int\_class1\_1F | ACACCGTGGAAACGGATGAA |
| Int\_class1\_2F | GATGAAGGCACGAACCCAGT |
| Int\_class1\_3F | GGAAACGGATGAAGGCACGA |
| T7\_Int\_class1\_2F | TAATACGACTCACTATAGGATGAAGGCACGAACCCAGT |
| Int\_class1\_1R | TCTAAGAGCGGCGCAATACG |
| Int\_class1\_2R | CGGTTGGAAGCTGTCGATTG |
| Int\_class1\_3R | CCTCGAAGAACCGCACAATC |

We successfully applied these primers to a control strain P. aeruginosa that contains a known Class I integron that contains VIM-2 metallo-beta-lactamase (“Characterization of a Bulgarian VIM-2 metallo-β-lactamase-producing Pseudomonas aeruginosa clinical isolate belonging to the
high-risk sequence type 111” doi.org/10.1080/23744235.2021.1934531).

**5.** We developed a novel T-cloning vector that allows efficient TA cloning of integron gene cassettes. The TA cloning module utilizes two restriction sites for the AhdI enzyme that generate single T-overhangs upon digestion. Its sequence was submitted to the Part registry, under number BBa\_K3764000 (Aleks link). On the following picture, you can see the results, of a controlled TA cloning, that uses the mRFP1 expression cassette as a test insert.

![image](  https://static.igem.org/mediawiki/2021/7/7f/T--Bulgaria--TA-cloning.jpg)

**6.** We optimized the design of the cloning vector with removing the LacZα fragment and putting the mRFP1 coding sequence in its place. This novel module was submitted to the Parts registry under the number BBa\_K3764001

**7.** We successfully transferred the blaVIM-2 containing gene cassette in E. coli, after cloning it in our T-vector based on module BBa\_K3764000

![image](  https://static.igem.org/mediawiki/2021/b/b6/T--Bulgaria--Mer.jpg)

**8.** We designed three primer pairs that can amplify the QRDR region of the gyrA gene in E. coli

| Name | Sequence |
| --- | --- |
| Primer F1 | GAGCGACCTTGCGAGAGAA |
| Primer F2 | ATGAGCGACCTTGCGAGAG |
| Primer F3 | AGCGACCTTGCGAGAGAAAT |
| Primer R1 | CGTTAATGATTGCCGCCGTC |
| Primer R2 | GCTTCTTCAATACCGCGACG |
| Primer R3 | GACCGTTAATGATTGCCGCC |

**9.** We developed a reporter vector that contains the remaining part of the gyrA coding sequence in combination with XbaI and HintIII restriction
sites, that allow in frame cloning of QRDR region PCR primers. Upon test with fragments that contain known mutations which confers quinolone resistance we did not observe colonies on petri dishes with levofloxacin. This was due to the dominant phenotype carried by the chromosomal gyrA gene copy.

**10.** We created a novel cloning-based system that can be used for monitoring the formation of unproductive chimera and artifacts between highly similar DNA sequences in PCR amplifications. We needed this tool because we wanted to see if the PCR amplification step of the QRDR regions found in complex samples can lead to chimera formation, that has the potential to bias our results. This system consists of two parts, that were submitted to the registry - BBa\_K3764002 and BBa\_K3764003 (aleks link?). They both consist of mRFP1 coding sequence but two tyrosine codons in them were mutated to amber (TAG) stop codons. We mixed both these parts and amplified the mixture before cloning it into a vector with constitutive promotor and RBS, results are presented on the following picture.

![image](  https://static.igem.org/mediawiki/2021/2/22/T--Bulgaria--Chimera.jpeg)

The presence of red colonies indicates chimera formation that can have influence on our findings. To limit this, we plan to use emulsion PCR that limits such events by physically separating the DNA matrix molecules.

**11.** We made a model for antibiotic resistance occurrence, using laboratory E. coli strain and sublethal concentrations of the antibiotic rifampicin. We used normal cultivation with fixed antibiotic concentrations as well as a continues growth with increasing rifampicin amounts. In both cases, we quickly observed the occurrence of resistant clones, as indicated by the antibiograms presented on the following photos:

![image](https://static.igem.org/mediawiki/2021/0/00/T--Bulgaria--KRXsens.jpg)

![image]( https://static.igem.org/mediawiki/2021/4/4f/T--Bulgaria--krxres.jpg)

Unmodified KRX antibiogram is on the left and the resultant resistant strain on the right.