iGEM 2022 — Team uni-hamburg

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May 2022

June 2022

July 2022

August 2022

September 2022

October 2022

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Antibiotic-resistant bacteria pose a growing threat in developing and industrial regions alike. While antibiotics are widely available, it often remains unclear which antibiotics the germs at hand are resistant against. Currently, there are two approaches for the treatment of resistant bacteria: Treatment with a panel of antibiotics, or targeted treatment based on an antibiogram. Treating with multiple antibiotics may give rise to additional resistances in already dangerous bacteria. An antibiogram takes at least 3 days before a clear result is available because of the slow bacterial growth, limiting time available for treatment.[1](#antibiogram)

### Design

We designed a point-of-care diagnostic method for detecting antibiotic resistances in patient samples. To accelerate diagnosis, we decided to directly detect the resistance genes instead of the bacteria’s response to antibiotics. A common challenge in synthetic biology is speed and adaptability of biosensors and genetic circuits. We aimed our solution to be adaptable to any resistance gene, and to reliably detect and report its presence in as little time as possible. To keep the degrees of uncertainty at a minimum, we decided to import our system into the bacteria in question, for example using phages. By regulating translation instead of transcription, we aimed to ensure adaptability to a wide panel of resistance genes, and rapid response to their presence. Recently, split ribozymes were published as a technology able to regulate translation in response to presence of a target RNA. Split ribozymes are programmable using a guide RNA to detect any RNA, which renders them a great fit for our project. We designed split ribozymes to detect mRNA of native and resistance genes. To report detected resistance mRNA, we designed multiple reporters: GFP, a fluorescent protein, eforRED, a chromoprotein and the enzyme horseradish peroxidase.

### Build

We built our constructs using GoldenGate Assembly (GGA). Our first design did not work because we tried to assemble too many small parts in one GGA. Therefore, we changed our strategy and ordered bigger parts to be synthesized that were much easier to assemble in one reaction. In order to get these synthesized, we had to change the promoters to be different but in a similar strength range. If the promoters were the same the region of similarity is too big to get synthesized.

### Test

As proof of concept, we introduced our system into  E. coli DH5alpha by transformation. In the finished application, we propose using phages. We confirmed that our GGAs worked with a colony PCR that showed mostly positive results. We measured fluorescence for GFP at an emission of 485 nm and excitation 515 nm as well as absorbance assays for the chromoprotein eforRED at a wavelength of 582 nm.

### Learn

Our system did not show any meaningful activity. We were only able to measure GFP in the inducible GFP positive control where GFP is controlled by a T7 and lacO promoter. We did not get a measurement for eforRED even for the positive control. The lack of signal can be explained by the relatively weak promoter strength of the chosen promoters BBa\_J23115 and BBa\_J23116. Our [model](https://2022.igem.wiki/uni-hamburg/model) showed that with stronger promoters like BBa\_J23101 and BBa\_J23104 the signal strength is much higher as well. We designed our system to have a low background signal but that resulted in us getting no signal at all. So in the next engineering cycle we will need to use the stronger promoters. For eforRED it is also important to mention that chromoproteins need time to get visible and we wanted to measure our samples promptly as our end goal is a point of care diagnostic device. So chromoproteins might not be the way to go for a quick and reliable reporter.

[1] <https://medmix.at/antibiogramm-antibiotika-einsatz/>

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## Our Protocols

---

Transformation

Here you can find the protocols we used for our transformations

 Standard transformation
Materials:

* DNA
* Distribution Kit
* Competent cells
* SOC Media

Steps:

1. Resuspend DNA in selected wells in the Distribution Kit with 10μl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
2. Label 1.5mL tubes with part name or well location. Fill the lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control).
3. Thaw competent cells on ice: This may take 10-20 min for a 260 μl stock.Dispose of unused competent cells. Do not refreeze unused thawed cells, asit will drastically reduce transformation efficiency.
4. Pipette 50 μL of competent cells into 1.5 mL tube: 50 μL in a 1.5 mL tube pertransformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.
5. Pipette 1 μL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice. Mix gently, e.g. by moving the tube over a tube rack.
6. Close 1.5 mL tubes, incubate on ice for 15 min.
7. Heat shock tubes at 42 °C for 45 s. Ensure the bottoms of the tubes are submerged. Timing is critical.
8. Incubate on ice for 5 min: Return transformation tubes to ice bucket.
9. Pipette 800 μL prewarmed SOC media (37 °C) to each transformation.
10. Incubate at 37 °C for 1 ho at 300 rpm.
11. Centrifuge  for 1 min at 4500 rpm, removement of the supernatant until 100 µL and suspend the pellet in remained medium. The cells are plated out onto (antibiotic) agar plates and incubated overnight at 37°C.

 Transformation with NEB Cells
Materials:

* DNA
* Distribution Kit
* Competent cells
* SOC Media

Steps:

1. Resuspend DNA in selected wells in the Distribution Kit with 10μl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
2. Label 1.5mL tubes with part name or well location. Fill the lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control).
3. Thaw competent cells on ice: This may take 10-20 min for a 260 μl stock.Dispose of unused competent cells. Do not refreeze unused thawed cells, asit will drastically reduce transformation efficiency.
4. Pipette 50 μL of competent cells into 1.5 mL tube: 50 μL in a 1.5 mL tube pertransformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.
5. Pipette 1 μL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice. Mix gently, e.g. by moving the tube over a tube rack.
6. Close 1.5 mL tubes, incubate on ice for 15 min.
7. Heat shock tubes at 42 °C for 45 s. Ensure the bottoms of the tubes are submerged. Timing is critical.
8. Incubate on ice for 5 min: Return transformation tubes to ice bucket.
9. Pipette 800 μL prewarmed SOC media (37 °C) to each transformation.
10. Incubate at 37 °C for 1 ho at 300 rpm.
11. Centrifuge  for 1 min at 4500 rpm, removement of the supernatant until 100 µL and suspend the pellet in remained medium. The cells are plated out onto (antibiotic) agar plates and incubated overnight at 37°C.

Colony PCR
Materials:

* Template DNA
* Forward Primer/ Reverse Primer
* Buffer
* 10 mM dNTPs
* Taq Polymerase
* LB-Agar plate

Steps:

1. Prepare the mastermix with the following reagents:

|  |  |
| --- | --- |
| Reagents | Volume |
| Buffer | 20 µL |
| 10 mM dNTPs | 2 µL |
| Taq Polymerase | 1 µL |
| Water | 65 µL |

2. Add 0.5 µL of each primer (forward and reverse primer) to 8.8 µL of Mastermix.
3. Add 0.2 µL of ORI to the corresponding tubes.
4. Pick three colonies from each plate.
5. Inoculate the labeled spot on an LB-Agar plate.
6. Shortly dip the rest of the colony into the PCR mix, then resuspend in LB-Medium with Camp for overnight cultures.
7. Transfer tube to a thermocycler with the following program:

|  |  |  |
| --- | --- | --- |
| Temperature (°C) | Time (mm:ss) | Cycle |
| 98 | 10:00 |  |
| 95 | 00:30 | 25 |
| xx | 00:30 | 25 |
| 72 | xx | 25 |
| 72 | 5:00 |  |

Gel extraction
Materials:

* agarose gel with DNA from gelelecktrophoresis
* Membrane Binding Solution
* SV Minicolumn
* Membrane Wash Solution
* Nuclease-free water

Steps:

1. Slice DNA out of the agarose gel and weigh the gel sclice.
2. Add Membrane Binding Solution at a ratio of 10 µL/10 mg of agarose gel slice.
3. Insert SV Minicolumn into the tube.
4. Incubate at 60 °C for 10 minutes or until the slice is completely dissolved and vortex every three minutes
5. Centrifuge at 16,000 x g for 1 minute, then discard the flowthrough and reinsert SV Minicolumn.
6. Add 700 µL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g for 1 minute,  then discard flowthrough and reinsert SV Minicolumn.
7. Add 400 µL Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes,  then discard flowthrough and reinsert SV Minicolumn.
8. Empty the tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol.
9. Transfer Minicolumn to clean 1.5 ml microcentrifuge tube.
10. Add 50 µL of Nuclease-Free Water to Minicolumn and incubate at room temperature for 1 minute. Centrifuge at 16,000 x g for 1 minute.
11. Discard Minicolumn and store DNA at -20°C.

Overnight cultures
Materials:

* 15 mL culture tube
* LB medium
* 1000X antibiotic stock solution
* Plate with colonies
* Micropipette tips

Steps:

1. Add 2-5 mLLB medium into a culture tube and add 1 uL of antibiotic stock solution per 1 mL of medium.
2. Pick one colony using a micropipette tip and eject it into the culture tube.
3. Incubate overnight at 37°C and 150 - 250 RPM.

PCR for part amplification
Materials:

* Template DNA with part
* Q5 High-Fidelity DNA-Polymerase
* 5XQ5 Reaction Buffer
* 10 mM dNTPs
* 10 uMFw-Primer
* 10 uMRw-Primer
* Nuclease-free water

Steps:

1. DNA dilution to 10 ng/µL
2. Primer dilution to 100 µM
3. Setup the PCR reaction mixes with each following the scheme:

|  |  |  |
| --- | --- | --- |
| Component | 50 uL Volume (same ratios for the 20 uL reactions) | Final concentration |
| 5x Q5 Reaction Buffer | 10 | 1x |
| 10 mM dNTPs | 1 | 200 uM |
| 10 uM Fw | 2,5 | 0,5 uM |
| 10 uM Rv | 2,5 | 0,5 uM |
| Template DNA (10 ng/uL) | 1 | 10 ng / 50 uL reaction mix |
| Q5 High-Fidelity DNA-Polymerase | 0,5 | 0,02 U/uL |
| Nuclease-free water | 32,5 |  |

4. Run PCR following the scheme:

|  |  |  |
| --- | --- | --- |
| step | temperature | time |
| Initial denaturation | 98°C | 30 seconds |
| 35 cycles | 98°C  69°C  72°C | 10 seconds  30 seconds  30 seconds/kb see |
| Final Extension | 72°C | 2 minutes |
| Hold | 4 °C |  |

Agarose-Gel electrophoresis
Materials:

* Agarose
* 1 x TAE buffer
* Gel chamber
* DNA
* 6 x loading dye
* DNA ladder
* SYBR Green
* Camera

Steps:

1. Completely dissolve agarose in an appropriate concentration (e.g. 1 % ) in TAE buffer by heating.
2. Pour the solution in a gel chamber using a fitting comb and wait for the gel to fully polymerize (up to an hour).
3. Mix at least 10 µL of the PCR products with loading dye and 1:10 SYBR Green.
4. Load the gel with the samples and the DNA ladder.
5. Run the samples at 100-120 V for 30-45 minutes.
6. Analyze and image the gel under a camera with an UV illuminator.

Competent cells
Materials:

* Cells
* Centrifuge
* LB-Medium
* Ca/Glycerol buffer (60 mM CaCl2, 10 mM PIPES, 150 mL glycerol, fill water up to 1 L, pH=7.0)
* Liquid nitrogen

Steps:

1. Grow the cells at 37 °C till OD= 0.2/0.4.
2. Chill the culture on ice for 5 min.
3. Collect cells by centrifugation at 6000 rpm at 4 °C for 10 min.
4. Gently resuspend the cells from 500 mL LB in 40 mL cold Ca/glycerol buffer on ice.
5. Incubate the cells on ice for  5~30 min.
6. Repeat step 3 and 4.
7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30 min (the longer, the better).
8. Collect celly by centrifugation at 6000 rpm at 4 °C for 10 min.
9. Resuspend cells from 500 mL LB in 6 mL Ca/glycerol buffer.
10. Aliquote 150 µL/tube.
11. Freeze in liquid nitrogen and store at -80°C.

Miniprep
Materials:

* Bacterial culture
* Centrifuge
* Buffer LYSIS LP
* Buffer RESUSPENSION
* Buffer NEUTRALIZATION
* Buffer Wash PA
* Buffer Wash PB
* Buffer Elution EP
* Collection Tube with Mini Filter

Steps:

1. Transfer the culture into 2 mL Eppis.
2. Centrifuge at maximum speed for 1 min to pellet the cells and remove the supernatant completely and discard.
3. Resuspend the pellet in 250 µL Buffer RESUSPENSION and mix by vortexing or pipetting up and down.
4. Add 250 µL Buffer LYSIS LP and mix by inverting the tube seven times.
5. Incubate at 20 °C for 5 min. Invert the tube few more times during incubation for improved lysis.
6. Add 350 µL Buffer NEUTRALIZATION and mix gently.
7. Centrifuge at maximum speed for 8 min to precipitate proteins and large chromosomal DNA.
8. Transfer the clarified supernatant to Mini Filter (orange) placed in a Collection Tube.
9. Centrifuge at 12000 rpm for 1 min, discard the filtrate and re-use the Collection Tube.
10. Add 500 µL Buffer WASH PA to the Mini Filter.
11. Centrifuge at 12000 rpm for 1 min, discard the filtrate and re-use the Collection Tube.
12. Add 700 µL Buffer WASH PB.
13. Centrifuge at 12000 rpm for 1 min, discard the filtrate and re-use the Collection Tube.
14. Centrifuge at maximum speed for 2 min to remove residual ethanol,
15. Place the Mini Filter in a new Elution Tube and add 20 µL water to the center of  the Mini Filter.
16. Incubate at 20 °C for 1 min.
17. Centrifuge at 8000 rpm for 1 min and discard the Mini Filter.
18. Purified plasmid DNA in the Elution Tube can be used immediately.
19. Store the DNA at -20 °C.

 Plate reader measurement
Materials:

* 96 well microplate
* Bacteria culture expressing a reporter detectable at a specific wavelength
* LB media
* Microplate reader

Steps:

1. Pipette 300 μL of each culture in the top row of the 96 well plate (under the clean bench). Pipette the same volume of LB media into an additional top row well.
2. Pipette 200 μL of LB media into the rows beneath for each dilution
3. Pipette the volume of culture from the top row into the row beneath corresponding to the desired dilution (e.g. 50 μL for a 1:5 dilution) and resuspend 2-3 times
4. Repeat Step 3 for each of the following dilutions
5. Discard 50 μL of the last dilution
6. Place the lid on the 96 well plate and place it in the microplate reader
7. Use an excitation wavelength and detect an emission wavelength corresponding to the used reporter

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Our point-of-care diagnostic gadget must be able to identify both resistant mRNA and bacterial RNA in order to be successful. We require a reporter that produces a signal that is, ideally, both instantly detectable and visible. Therefore, we designed two split ribozymes [1](#splitribozyme) to detect either the stable endogenous *E. coli* mRNA hcat [2](#hcat) and the kanamycin resistance (KanR) of pSB1K3. We combined those with two different reporters: the fluorescent GFP and the chromoprotein eforRED.

We assembled our constructs with Golden Gate Assembly (GGA). To check if our GGAs worked correctly we performed a colony PCR. The reporter and the entire functioning split ribozyme unit make up the amplicon. It is 1700 bp long with GFP and 1661bp long with eforRED. There were a significant number of positive clones in the agarose gels. Figure 1 displays the gel of the clones that were further processed.

![](https://static.igem.wiki/teams/4472/wiki/parts/results/split-ribozym-colony-pcr-wiki.png)

Figure 1: agarose gel of colony PCR of our GGA constructs. Loading scheme: 1=1kb benchtop ladder, 2= split ribozyme hcat with GFP, 3= split ribozyme hcat with eforRED, 4= split ribozyme kan with GFP, 5= split ribozyme kan with eforRED. All in Backbone pSB1C3, top and bottom are each a clone of the same GGA

The positive clones were further tested using fluorescence and absorbance measurement in a plate reader. After 6 hours of culturing at 37°C, the bacteria were pipetted into 96-well plates. The plates for fluorescence measurement were black with a transparent bottom to minimize crosstalk. For absorbance measurements we used transparent 96 well plates. The bacteria were spun down, the pellet washed in water twice, then the bacteria were resuspended in water and transferred to the plate where a 1:5 serial dilution was performed. The absorbance of eforRED was measured at 230 nm and 582 nm. At 230 nm the platereader only gave values that were over the threshold of detection. At 582 nm, there was no difference between samples with eforRED and without, no signal was detectable. The kanamycin backbone was giving us pink colonies where there should not have been any (figure 2), therefore we were unable to measure the split ribozyme recognizing kanamycin resistance.

![](https://static.igem.wiki/teams/4472/wiki/parts/results/psb1k3-pink.jpg)

Figure 2: colonies of pSB1K3 with hcat split ribozyme and GFP

The fluorescence of GFP was measured at 485 nm emission and 515 nm excitation. The platereader read a significant amount of the emission laser at 510 nm for the excitation detection, rendering the data useless because all samples—including the negative control—had high values. At 515 nm we could detect just a very small difference between the negative control containing bacteria lacking GFP and the bacteria carrying our split ribozyme GFP construct. The positive control of just the one promoter BBa\_J23115 in front of the GFP is very low as well. This shows that the promoter is insufficient to provide a strong signal. The model supported this hypothesis.

![](https://static.igem.wiki/teams/4472/wiki/parts/results/gfp-weak-promoter.png)

Figure 3: Fluorescence measurement of GFP at emission 485 nm and excitation 510 nm normalized against the negative control (bacteria without GFP construct). Dilution indicated in legend.

In contrast to our split ribozyme constructs with GFP having almost no signal our construct with an inducible GFP provided a strong signal even when uninduced as displayed in figure 4.

![](https://static.igem.wiki/teams/4472/wiki/parts/results/gfp-uninduced.png)

Figure 4: Uninduced measurement of T7+LacO Promoter construct with GFP normalized against the negative control (bacteria without GFP construct)

## Conclusion and Outlook

To produce a point-of-care diagnostic device the split ribozymes need to be transcribed at a higher rate with stronger promoters. Our Model indicated that BBa\_J23101 and BBa\_J23104 are good candidates. Alternative reporters should be taken into account, as eforRED requires more time to get visible and GFP measurement is limited by a high background fluorescence and the emission laser. For the implementation we also need to consider how to get our system into the bacterium. Phages are helpful for the delivery due to their easy storage and long shelf life.  [3](#phage) There is still a lot to do, but we are determined to make it happen!

[1] [Gambill L, Staubus A, Ameruoso A, Chappell J. A split ribozyme that links detection of a native RNA to orthogonal protein outputs. bioRxiv. January 2022:2022.01.12.476080. doi:10.1101/2022.01.12.476080](https://www.biorxiv.org/content/10.1101/2022.01.12.476080v1)
[2][Zhou, K., Zhou, L., Lim, Q.'. et al. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Molecular Biol 12, 18 (2011). https://doi.org/10.1186/1471-2199-12-18](https://bmcmolbiol.biomedcentral.com/articles/10.1186/1471-2199-12-18#citeas)
[3][Yajie Zhang, Xiujuan Peng, Hairui Zhang, Alan B. Watts, Debadyuti Ghosh, Manufacturing and ambient stability of shelf freeze dried bacteriophage powder formulations, International Journal of Pharmaceutics, Volume 542, Issues 1–2, 2018, Pages 1-7, ISSN 0378-5173.](https://doi.org/10.1016/j.ijpharm.2018.02.023)

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