Chemical	Manufacturer
MilliQ water Q-Pod®	Merck
DpnI	Jena Bioscience
BamHI	Jena Bioscience
SalI	Jena Bioscience
NheI	Jena Bioscience
AseI	NEB
10X rCutsmart Buffer	NEB
10X Universal Buffer	Jena Bioscience
NEB HiFi Assembly Master Mix	NEB
NEB positive control reaction mix	NEB
Agar	Fluka Chemie AG
LB (Luria/Miller) – Broth	Carl Roth
Kanamycin (50 mg/mL)	Carl Roth
Ampicillin (100 mg/mL)	Carl Roth
SOC Outgrowth Medium	NEB
10X GC Buffer	NEB
10mM dNTPs	PCR Biosystems
DMSO	NEB
Phusion-Polymerase	kind gift by Prof. Dr. Jan Faix
96 – 100% Ethanol	Carl Roth
Glycerol	Sigma-Aldrich
DMEM, high glucose	Sigma-Aldrich
FCS	Sigma-Aldrich
Pen/Strep (Penicillin and Streptomycin)	Sigma-Aldrich
0.25% Trypsin/EDTA	Gibco
DPBS	Sigma-Aldrich
GenJet™ Reagent (Ver. II)	SignaGen Laboratories
CuSO4	Carl Roth
1M HEPES	Carl Roth
FACS-Buffer (DPBS + 1%FCS)	Self-made

Consumable	Manufacturer
Pipets	Eppendorf
Pipet Tips	Sarstedt
Serological Pipets	Sarstedt
1.5mL Reaction Tubes	Sarstedt
2mL Reaction Tubes	Sarstedt
PCR-Tubes	Sarstedt
Cryo-Tubes	Sarstedt
15mL Canonical Tubes	Sarstedt
50mL Canocical Tubes	Sarstedt
Cell Culture Plates	Sarstedt
Petri Dishes, 100mm	Sarstedt
Zyppy™ Plasmid Miniprep Kit	Zymo Research
ZymoPURE II Plasmid Midiprep Kit	Zymo Research
HEK293T cells	DSMZ
NEB competent E. coli DH5-alpha cells	NEB

A PCR can serve, on the one hand, as a preparative tool for amplification of individual fragments and, on the other hand, as an analytical tool to validate the reactions performed.

Component	50 µL Reaction	Final Concentration
10X GC Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
10 µM Forward Primer	2.5 µL	0.5 µM
10 µM Reverse Primer	2.5 µL	0.5 µM
DMSO (optional)	(1.5 µL)	3%
Phusion DNA Polymerase	0.5 µL	1.0 Units/50 µL PCR
Template DNA	1 µL	< 250 ng
Nuclease-free water	to 50 µL

Step	Time	Temperature	Cycles
Initial denaturation	30 sec	98°C	1
Denaturation	5-10 sec	98°C	30
Annealing*	10-30 sec	50-72°C	30
Extension*	20-30 sec/kb	72°C	30
Final extension	2 min	72°C	1

*The annealing temperature and extension time depends on primer use and product length, respectively.

Gene/Element	Sequence 5´->3´
HGm_MRE-prom_fw	CCGCCATGCATTAGTTATGCACACTGGCGCT
HGm_MRE-prom_rev	TGGCGACCGGTAGCGGACGCTTAGAGGACAGC
MTF-1_fw	CAGAGCTGGTTTAGTGAACCGTCAGATCCGATGGGGGAACACAGTCCAGAC
MTF-1_rev	gcccttagacaccatGGGTGGCAGCTGCAGG
mRuby2_fw	CTGCAGCTGCCACCCatggtgtctaagggcgaagagc
mRuby2_rev	ATCCCGGGCCCGCGGTACCGTCGACTGCAGcttgtacagctcgtccatccc
MTF-1_solo_fw	CAGAGCTGGTTTAGTGAACCGTCAGATCCGATGGGGGAACACAGTCCAGAC
MTF-1_solo_rev	ATCCCGGGCCCGCGGTACCGTCGACTGCAGCTAGGGTGGCAGCTGCAG
Atf-2_Fragment1_fw	TGAACCGTCAGATCCGatgaaattcaagttacatgtgaattctgccag
Atf-2_Fragment1_rev	ggatccccacttcctgagggctgtgac
Atf-2_Fragment2_fw	caggaagtggggatccaccggtcg
Atf-2_Fragment2_rev	TCAGTTATCTAGATCCGGTGcttgtacagctcgtccatccc
ccpA_Fragment1_fw	tggatccccttttgtagttcctcggtattcaattctgtgag
ccpA_Fragment1_rev	TGAACCGTCAGATCCGatgacagttactatatatgatgtagcaagagaagc
ccpA_Fragment2_fw	actacaaaaggggatccaccggtcg
ccpA_Fragment2_rev	TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc
graR_Fragment1_fw	TGAACCGTCAGATCCGatgcaaatactactagtagaagatgacaatactttgt
graR_Fragment1_rev	tggatccccttcatgagccatatatccttttcctacttttgt
graR_Fragment2_fw	ctcatgaaggggatccaccggtcg
graR_Fragment2_rev	TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc
pknB_Fragment_fw	AGCTTCGAATTCTGCAGAatgataggtaaaataataaatgaacgatataaaattgtagataagcttgg
pknB_Fragment_rev	TCAGTTATCTAGATCCGGTGttatacatcatcatagctgacttctttttcagctacag
eGFP-C_fw	GTCCTGCTGGAGTTCGTG
eGFP-N_fw	CAACGGGACTTTCCAAAATG
SV40_rev	cctctgcataaataaaaaaaattagtcagccatgg
H2A+ linker_Cla+PCR_rev	TCCTCGCCCTTGCTCACCATggtggcgaccgg
CMV_fw	CGCAAATGGGCGGTAGGCGTG
eGFP-C_rev	AGCTGCAATAAACAAGTT
eGFP-C1-anti	GGTTCAGGGGGAGGTGTG
SV40-pA_rev	CCTCTACAAATGTGGTATGG
ATF2_miRFP_BB_rev	cgatacaccatcacccggtcg
TVBB_ColE1_Ori_rev	ttcgccacctctgacttgag

Beside the amplification of our parts, the restriction digest of the backbone EGFP-C2 was performed. The digested backbone is needed to bring the parts in the whole construct together. Different restriction enzymes are used, depending on where the backbone should be cut and whether the already present EGFP is needed or should be replaced by mRuby or miRFP.

Component	50 µL Reaction	Final Concentration
10X rCUTSMART Buffer/10X UB Buffer	5 µL	1X
RE1	1 µL	20 units/50 µL
RE2	1 µL	20 units/50 µL
Backbone DNA	1 µg
Nuclease-free water	Add to 50 µL

The reaction is incubated at 37°C for one hour. To deactivate the enzyme, the mixture is incubated at 80°C for 20 minutes.

Plasmids	RE1	RE2
PknB EGFP C2	SalⅠ	BamHⅠ
GraR mRuby2 C2	NheⅠ	BamHⅠ
CcpA mRuby2 C2	NheⅠ	BamHⅠ
MTF-1 mRuby2 C2	NheⅠ	BamHⅠ
ATF2 mRuby2 C2	NheⅠ	BamHⅠ
ATF2 miRFP670 Promoter	AseⅠ	BamHⅠ
MRE EGFP Promotor	AseⅠ	NheⅠ

The DpnⅠ digestion is applied after the last PCR. The enzyme DpnⅠ belongs to type Ⅱ restriction endonucleases, which cleave at the methylated recognition sequence 5'-GmATC-3' in double-stranded DNA Text. This ensures that the remaining template is destroyed, as the desired PCR product is unmethylated. The procedure is analogue to the previous restriction digest.

Component	50 µL Reaction	Final Concentration
10X UB Buffer	5 µL	1X
DpnⅠ	1 µL	20 units/50 µL
Backbone DNA	1 µg

The reaction is incubated at 37°C for one hour. To deactivate the enzyme, the mixture is incubated at 80°C for 20 minutes.

Finally, the HiFi DNA Assembly, a further developed version of the Gibson assembly, which was created by Daniel G. Gibson^[2], is performed. It describes a cloning method, where multiple DNA fragments are joined together in a single reaction, regardless of size of DNA fragments and their overlaps. First, the overlapping 5´ ends of the DNA fragments are digested by an exonuclease and the 3´overhangs facilitate the annealing of the fragments at their overlapping regions. After that, a DNA polymerase extends the 3´ends by filling the gaps with dNTPs and lastly, a DNA ligase seals the nicks, so that a new double-stranded DNA construct emergesTextText. The protocol is shown in the next steps.

Insert & Vector preparation:

1. Inserts and vectors were amplified with overhang primers and verified according to the PCR protocol or were ordered as GeneBlocks with overhangs.

Assembly reaction:

1. Set up the following reaction on ice:
   

   Component	2–3 Fragment Assembly
   Recommended DNA Molar Ratio	vector:insert = 1:2
   Total Amount of Fragments	X µL = 100 ng vector : 200 ng inserts *
   NEBuilderHiFi DNA Assembly Master Mix	10 µL
   Deionized H2O	10-X µL
   Total Volume	20 µL

   *Volume of fragments depends on measured concentration.

2. Incubate samples in a thermocycler at 50°C for 1 hour. For following incubation, store samples on ice or at –20°C for subsequent transformation.
3. Transform competent E. coli DH5 alpha (NEB) cells with 2 μl of the chilled assembled product, following the transformation protocol (NEB).

The ready to use plasmids are transferred in Competent E. coli DH5-alpha cells via heatshock transformation. The plating of the transformed cells on LB-Agar plates containing the antibiotic Kanamycin ensures that just positive cells, containing the plasmid with the Kanamycin resistance cassette, can grow.

Agar plate preparation:
Three plates of Kanamycin (50 µg/mL) containing LB-Agar plates (1.5% Agar) were prepared before starting the transformation protocol. For this, approximately 15 mL is used per plate. Two plates are for plating the bacteria, one is used as negative control and subsequent master plate.

Protocol:
Competent NEB®5-alpha E. coli bacteria, which were kindly sponsored by New England Biolabs, were used according to manufacturer´s protocol to introduce the cloned plasmids into bacteria for propagation of the expression vectors. For this purpose, 50 µL of NEB®5-alpha E. coli was mixed with 2 µL of the respective vector and incubated on ice for 30 minutes. Thereafter, heat shock was performed at 42°C for exactly 45 seconds. For recovery, the bacteria were placed on ice for a few minutes and 250 µL of SOC Outgrowth Medium (room temperature) was added. In this medium, the bacteria were shaken at 37°C and 180 rpm for one hour and then spread on agar plates containing kanamycin (50 µg/mL). Overnight, the bacteria incubated at 37°C and were selected by the antibiotic.

By picking the grown colonies from the agar plates, colony PCRs can be performed. Via this method, the correctness of the inserts can be validated and if the clones can be used for the next steps. For the colony PCR are different primers used than for the amplification.

Colony-PCR each with 20 µl preparations with for 10 clones each:

1. Colony PCR and master plate preparation.
   
   • Prepare 10x 1.5 mL reaction tubes for each cloning approach by adding 20µl ddH₂O into each tube.
   • Pick a nicely grown clone without visible satellite colonies using a sterile pipette tip, transfer a small amount of bacterial colony to master plate by tipping onto prepared and sufficiently labeled agar plate, and place tip – still containing bacteria – into ddH₂O within prepared 1.5 mL tube.
   • Repeat previous step 9x, so that there will be ten clones to be analyzed in total.
   • Transfer 5µl ddH₂O including bacteria into a PCR reaction tube for each clone.
   • Incubate PCR reaction tubes for 10 min at 80 °C. This step causes lysis of the dissolved bacteria, thereby releasing potential plasmid DNA from within the cells and facilitating the use of this preparation as template for the following analytical PCR reaction.
     
     
2. Prepare Master-mix.
   
   

   Component	Volume [µL] (other constructs)	Volume [µL] (PknB)
   10X GC-Puffer	2.5	4
   10 mM dNTPs	0.25	0.4
   10µM Forward Primer	0.625	1
   10 µM Reverse Primer	0.625	1
   Phusion-Polymerase	0.125	0.2
   Template	5	5
   DMSO (optional)	0.375	0.6
   ddH2O	Fill up to 12.5	Fill up to 20
   Total volume	12.5	20

3. Run the respective PCR program in the thermal cycler.
   
   

   Step	Time	Temperature	Cycles
   Initial denaturation	30 sec	98°C	1
   Denaturation	5-10 sec	98°C	30
   Annealing	10-30 sec	50-72°C	30
   Extension	20-30 sec/kb	72°C	30
   Final extension	2 min	72°C	1

   The annealing temperature and extension time depends on primer use and product length, respectively.
   
   

   Construct/Vector	Annealing Temp	Elongation time	Used for primer	Used rev Primer	DMSO
   M1.1	72°C	15 sec	HMG-MRE-Promotor_fw	H2a+linker Cla+PCR_rev	+
   M1.2	72°C	15 sec	HMG-MRE-Promotor_fw	H2a+linker Cla+PCR_rev	+
   M1.3	72°C	15 sec	HMG-MRE-Promotor_fw	H2a+linker Cla+PCR_rev	+
   M1.4	72°C	15 sec	HMG-MRE-Promotor_fw	H2a+linker Cla+PCR_rev	+
   M3.0	56°C	1 min	eGFP-N_fw	eGFP-C_rev	-
   A1	65°C	30 sec	eGFP-N_fw	eGFP-C_rev	-
   A2	65°C	30 sec	eGFP-N_fw	eGFP-C_rev	-
   A3	63°C	1 min	eGFP-C_fw	eGFP-C_anti	+
   A4	68°C	1 min	CMV_fw	SV40_rev	-
   A5	65°C	30 sec	ATF2_miRFP_BB_rev	TVBB_ColE1_Ori_rev	-

After the validation of the correct insert via colony PCR, the plasmid preparation was performed. The Mini and Midi Preps are necessary to extract and purify the plasmids. Finally, the plasmids are sequenced by Microsynth SeqLab to validate the purified DNA sequences so there are no mutations introduced through the replication.

Overnight Culture:

1. 5 ml LB
   
2. Add 5 µl of kanamycin from a 50 mg/mL stock solution to achieve a final concentration of 50 µg/mL.
3. Use the remaining 15 µL of the previously prepared solution of the picked bacterial clone within ddH₂O (see section colony PCR) to inoculate the overnight culture.
   Incubate the culture at 37 °C with shaking at 180 rpm overnight.

Glycerol Stock:

1. Prepare a 50% bacterial glycerol stock solution by mixing 500 µL of 100% glycerol with 500 µL of the overnight bacterial culture.
2. Store the glycerol stock at -80 °C.

Plasmid Preps:
All plasmid preps were conducted using Mini and Midi Prep Kits, generously sponsored by Zymo Research, according to the manufacturer’s protocol.

For the Plasmid Midi Prep a 5 mL LB + Kan pre-culture was prepared (according to the overnight culture section) and is incubated for 3-4 hours. 1 mL from the pre-culture was used to inoculate 150 mL of fresh LB medium + 150 µL kanamycin and was incubated overnight at. The next day, 1:1000 chloramphenicol was added to the overnight culture and incubated for another one hour before prepping.

Plasmid Validation via Sequencing:
Use 1 µg of plasmid DNA after elution and add nuclease-free water to 12 µL total volume.

For running culture (on Monday & Friday) and transfection culture (18 to 24 hours before transfection):

1. Pre-heat DPBS and DMEM with 10% FCS and 1% Pen/Strep or without Pen/Strep in water bath at 37°C.
2. Remove media from cells.
3. Wash cells with 5 mL DPBS.
4. Detach cells with 0.25% Trypsin+EDTA (2mL) and incubate for 5 min at 37°C.
5. Add double volume of medium (4mL) to stop reaction.
6. Centrifuge at 500 rcf for 5 min.
7. Discard supernatant and resuspend pellet in 1 mL medium.
8. Cell counting:
   
   1.  Dilute cells 1:50.
   2.  Prepare Neubauer chamber and add approx. 10 µL of diluted cells.
   3.  Count the 4x4 fields four times and calculated mean value.
9. Formulas to calculate your cell number and number of cells to seed:
   

   

   

10. Use for running culture 100mm Petri Dish or 24/48 Well plate.
    
    1.  Use 10mL of medium and transfer calculated volume of cell suspension.
    2.  Use 300µL for 48 Well plates or 500µL for 24 Well plates and transfer calculated volume of cell suspension.
11. Incubate at 37°C with 5% CO_2.

Transfection is used to insert and express foreign DNA, like our designed plasmids, into eukaryotic cellsText. For this, we use GenJet™ In Vitro DNA Transfection Reagent (Ver. II). A transfection efficiency of about 80 to 90% should be reached in HEK-293T cellsText. The transfection is proceeded by following the protocol of SignaGen® Laboratories.

Culture Dish	Culture Medium [mL]	Plasmid DNA [µg]	Diluent Volume [mL]	GenJet™ Reagent [µL]
48 Well Plate	0.3	0.25	2 x 0.015	0.75
24 Well Plate	0.5	0.5	2 x 0.025	1.5
6 Well Plate	2	1	2 x 0.05	3

• Part 1 – Cell Seeding:
  
  1.  Plate the cells 18 to 24 hours before transfection.
  2.  A confluency of 70 to 80% is recommended.
  3.  Change medium 30 to 60 minutes before transfection (Use DMEM High Glucose with 10% FCS and 1% Pen/Strep or without Pen/Strep).
• 
  Part 2 – Preparation of GenJet^-DNA Complex and Transfection Procedures:
  
  1. The recommended GenJet™ (µL)-DNA (µg) ratio of 3:1 (Table 13) is used.
  2. Use serum-free DMEM with High Glucose with 1% Pen/Strep or without Pen/Strep to dilute the GenJet^ Reagent and the plasmid DNA.

     
     The following steps depending on culture dish size (Table 13):

  3. Add X mL fresh DMEM with 10% FCS and 1% Pen/Strep or without Pen/Strep 30-60 min before transfection.
  4. Dilute X µg DNA to X mL serum-free DMEM with High Glucose with 1% Pen/Strep or without Pen/Strep; diluent volume -> mix by gently vortexing.
  5. Dilute X µL GenJet™ Reagent into X mL serum-free DMEM with High Glucose with 1% Pen/Strep or without Pen/Strep; diluent volume -> mix by pipetting 3-4 times.
  6. Transfer diluted GenJet™ Reagent to diluted DNA and mix by pipetting 3-4 times and incubate for approx. 10 mins at room temperature to form the GenJet™ -DNA Complex.
  7. Add the GenJet™-DNA Complex drop wise onto the media in each well and swirl gently.
  8. Change medium after 12 to 18 hours.
  
  Note: for transfection with two constructs divide the total amount of DNA and use equal amounts of each plasmid DNA (µg) (Table 13).
  -> volume of GenJet™ Reagent does not change

After successful transfection, the HEK293T cells should be able to indicate the presence of copper sulphate and ampicilin. Different concentrations were used to stimulate the expression of the fluorescent proteins and thus the detection of metals and antibiotics. The stimulation protocol is shown below.

CuSO₄:
The metal solutions were added after 24 h after of transfection. Before stimulation, the transfected cells were imaged to check the basal fluorescence signal. Moreover, 10 mM HEPES was added to fresh medium. The bivalent ions getting in the cells and bind to the MTF-1, which induces the MRE-containing promoters, which leads to eGFP expression. The maximal concentration of 2 mM, for CuSO₄, is according to WHO guidelines.

Concentration viability test [µM]	Concentration for induction [µM]
2000	500
1000	300
500	250
250	200
50	100
0	50
	0

Ampicillin:
Ampicillin was added after 24 h after transfection. Before the stimulation, the transfected cells were imaged to check the basal fluorescence signal before and after. The PASTA domain of PknB recognize beta-lactams, leading to phosphorylation of ATF2, which then binds to the AP1 and CRE sites in the promoter. The binding leads to the expression of miRFP670.

Concentration viability test [µg/mL]	Concentration for induction [µg/mL]
500	100
100	25
50	10
25	5
5	2.5
0	0

Before stimulation and after an incubation time of 4 hours, the cells were imaged by confocal microscopy. By this, we can qualitatively validate the reporter gene expression and localization of our membrane protein (PknB) and transcription factors.

To check the localization and expression of the proteins, confocal microscopy is used. Confocal microscopy is an advanced optical imaging technique that enhances the resolution and contrast of micrographs by using a spatial pinhole to block out-of-focus light. Unlike conventional wide-field microscopy, which captures light from all focal planes, confocal microscopy focuses on a single plane at a time, producing high-resolution, three-dimensional images. This is achieved by scanning the specimen point by point with a laser and collecting the emitted light through a pinhole that aligns with the focus plane. The result is sharp images with minimal background noise, making confocal microscopy ideal for detailed studies of cells, tissues, and other biological specimens. The images were recorded with a 25x/0.9 water-immersion objective. For the detection of the fluorescent signal, lasers with wavelengths of 488nm, 543nm & 630nm were used. The pictures were recorded with a frame average of four and a format of 1014 x 1024 pixel.

Later, the pictures were taken with a Leica camera system. For this, channels for eGFP (488 nm), mRuby (543 nm) and miRFP670 (630 nm) were chosen to validate the fluorescent signal. For brightfield recordings exposure times of 1 ms are used, for eGFP up to 70 ms and for mRuby between 100 ms and 1000 ms depending on signal strenght.
 
For both setups a pinhole of 58 µM and 75 µM was used to adjust the brightness. In general, all setup points depending on the sample and the fluorescent signal.
 
The pictures were analyzed via the Fiji (ImageJ) software.

Beside the qualitative validation of the gene expression, we want to quantify the expression level of the promoter coupled reporter genes. FACS analysis is an appropriate method to verify the functionality of the bio sensors.

Fluorescent Activated Cell Sorting (FACS) is a specialized type of flow cytometry that allows sorting and analysis of cells based on specific characteristics. Cells are first labeled with eGFP (metal promoter) or miRFP670 (antibiotic promoter). As the cells pass through a laser beam in a fluid stream, they emit light at different wavelengths depending on the markers present. In our experiments we use lasers with wavelengths of 488 nm (blue) and 635 nm (red). Detectors capture this light, and the cells are then sorted into different containers based on the fluorescent signals they emit. We use this technique to quantify our promoter activities with and without metal or antibiotic induction.

Protocol:

1. Discard media and wash with 300 µL DPBS.
   
   •  Discard DPBS.
2. Add 150 µL 0.25% Trypsin-EDTA.
   
   •  Incubate at 37°C for 5 min.
3. Stop reaction with 300 µL DMEM with 10% FCS & 1% Pen/Strep and transfer volume to FACS-Tubes.
4. Centrifuge at 800 rcf for 5 min.
5. Discard flow-through.
   
   •  Resuspend pellet in 1 mL FACS-Buffer.
   •  Repeat step 4.
6. Resuspend pellet in FACS-Buffer (volume depends on pellet size and cell number).
7. FACS measurement.
8. Analysis with FCSalyzer software and Microsoft Excel.