Plasmid piG_01a (BBa_K4604016) consists of an accidentally leaky version of the tet-promoter, which includes the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059), that can be induced with doxycycline. Additionally, it contains bluB (BBa_K4604005), the gene responsible for AdoCbl production, and the rrnB terminator (BBa_K4604003). The backbone used for cloning was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance.

Cloning Strategy

The tet promoter and rrnB terminator were provided to us by iGEM Freiburg 2022. We utilized one of their plasmids as a template for PCR but performed site-directed mutagenesis on the tet promoter to conform to iGEM parts standards. The bluB gene was synthesized by IDT, and its sequence was sourced from the National Library of Medicine (gene ID: 61603312) from Sinorhizobium meliloti 2011.
For the PCR, we followed the general protocol for the Q5 polymerase with varying parameters such as elongation time and annealing temperature:

The resulting PCR products were loaded onto an agarose gel with a DNA ladder. The correct bands were then cut out and extracted. Subsequently, the parts were assembled using the Golden Gate cloning method following the general protocol. After transformation, the resulting colonies were screened by colony PCR following an approximate 12-14 hour incubation period.
DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to verify correct insertion and absence of mutations.

Plasmid piG_01b (BBa_K4604015) consists of the tet promoter (comprising the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059)), which can be induced with doxycycline, bluB (BBa_K4604005), the gene responsible for AdoCbl production, and the rrnB terminator (BBa_K4604003). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance. Plasmid piG_01b is identical to piG_01a, except for a shorter region between the tet operator and the ribosome binding site (before approximately 50 bp, after approximately 20 bp).

Cloning Strategy

Plasmid piG_01a (BBa_K4604016) served as the template. For the PCR, we followed the general protocol for the Q5 polymerase with an annealing temperature of 56°C and an elongation time of 5 minutes. Subsequently, a Dpn1 digest was conducted at 37°C for an hour. The resulting DNA was loaded onto a 1% agarose gel with a DNA ladder, and the correct bands were then excised and extracted.
The parts were assembled using the Golden Gate cloning method in accordance with the general protocol. Following transformation, the resulting colonies were screened by colony PCR after an approximate 12-14 hour incubation period. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to verify correct insertion and absence of mutations.

Plasmid piG_02a (BBa_K4604017) comprises an accidentally leaky version of the tet-promoter (BBa_K4604002) and tet operator (BBa_K4229059), inducible with doxycycline, a riboswitch for AdoCbl (BBa_K4604031), mazF (toxin, BBa_K4604011), mTurquoise (BBa_K4604013), and the rrnB terminator (BBa_K4604003). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance.

Cloning Strategy

The tet promoter and rrnB terminator were provided by iGEM Freiburg 2022. We used one of their plasmids as a template for PCR but performed site-directed mutagenesis on the tet promoter to comply with iGEM parts standards. MazF (toxin), along with the riboswitch, was synthesized by IDT, with the sequence taken from the National Library of Medicine, specifically from E. coli K12 MG1655 (gene ID: 947252). The gene for the fluorescent protein mTurquoise was obtained from a plasmid by iGEM Freiburg 2022 (BBa_K4229064).
For the PCR, we followed the general protocol for the Q5 polymerase with varying parameters such as elongation time and annealing temperature:

The resulting PCR products were loaded onto an agarose gel with a DNA ladder, and the correct bands were cut out and extracted. The parts were then assembled using the Golden Gate cloning method following the general protocol. After transformation, the resulting colonies were screened via colony PCR after an approximate 12-14 hour incubation period.
For potential colonies containing the insert, an overnight culture (5 mL LB-medium, 34 mg/mL chloramphenicol) was prepared, and the DNA was isolated after incubation. The resulting plasmid was sent for sequencing to verify correct insertion and ensure no mutation.

Plasmid piG_02b (BBa_K4604018) comprises the tet promoter (consisting of the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059)), inducible with doxycycline, a riboswitch for AdoCbl (BBa_K4604031), mazF (toxin, BBa_K4604011), mTurquoise (BBa_K4604013), and the rrnB terminator (BBa_K4604003). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance. PiG_02b is identical to piG_02a, except for a shorter region between the tet operator and the ribosome binding site (before approximately 50 bp, after approximately 20 bp).

Cloning Strategy

Plasmid piG_02a (BBa_K4604017) served as the template. For the PCR, we followed the general protocol for the Q5 polymerase with an annealing temperature of 56°C and an elongation time of 5 minutes. Subsequently, a Dpn1 digest was conducted at 37°C for an hour. Afterward, the DNA was loaded onto a 1% agarose gel, and the correct bands were cut out and extracted. Gibson Assembly was employed according to the protocol to assemble the plasmid. Following transformation, the resulting colonies were screened by colony PCR after an approximate 12-14 hour incubation period.
For potential colonies containing the insert, DNA was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and ensure no mutation.

Plasmid piG_03 (BBa_K4604019) is composed of the tet promoter (consisting of the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059)), inducible with doxycycline, a riboswitch for AdoCbl (BBa_K4604031), mazF (toxin, BBa_K4604011), and the rrnB terminator (BBa_K4604003). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance. Plasmid piG_03 is identical to piG_02b, excluding mTurquoise (fluorescent marker protein).

Cloning Strategy

Plasmid piG_02b (BBa_K4604018) served as the template. For the PCR, we followed the general protocol for the Q5 polymerase with an annealing temperature of 62°C and an elongation time of 2 minutes. Subsequently, a Dpn1 digest was conducted at 37°C for an hour. Afterward, the DNA was loaded onto a 1% agarose gel, and the correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. Following transformation, the resulting colonies were screened by colony PCR after an approximate 12-14 hour incubation period.
For potential colonies containing the insert, DNA was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and ensure no mutation.

Plasmid piG_07 (BBa_K4604020) is composed of the tet promoter (consisting of the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059)), inducible with doxycycline, a mutated version of bluB (BBa_K4604006), the gene responsible for AdoCbl production, and the rrnB terminator (BBa_K4604003). The mutation introduced a stop codon, terminating translation prematurely. The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance.

Cloning Strategy

Plasmid piG_01b (BBa_K4604015) served as the template. For the PCR, we followed the general protocol for the Q5 polymerase with an annealing temperature of 63°C and an elongation time of 2.5 minutes. Subsequently, a Dpn1 digest was carried out at 37°C for 2 hours. Afterward, the DNA was loaded onto a 1% agarose gel, and the correct bands were cut out and extracted. The linear fragment was directly used for transformation, which was performed according to the general protocol. The resulting colonies after an approximate 12-14 hour incubation period were screened by colony PCR.
DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and ensure no mutation.

Plasmid piG_08 (BBa_K4604035) is composed of the constitutive promoter of ampicillin resistance (BBa_K2308010), mazE (antitoxin, BBa_K4604037), and the rrnB terminator (BBa_K4604036). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance.

Cloning Strategy

The amp promoter, as well as the rrnB terminator, were used for cloning from a plasmid of the iGEM 2022 Freiburg team. The mazE gene was synthesized by IDT, with the sequence taken from the National Library of Medicine (gene ID: 947245) from E. coli K12 MG1655. For the PCR, we followed the general protocol for the Q5 polymerase with varying parameters for elongation time and annealing temperature:

Since regular Golden Gate cloning did not work with the plasmid, we adopted a different approach. We fused the individual parts (as shown in the table above) together via PCR to create one large fragment. Complementary overhangs were designed and attached to the primers used to amplify the individual fragments. First, we fused the amp promoter with mazE, and then added the terminator. This insert was amplified according to the Q5 protocol with a 62°C annealing temperature and an elongation time of 1 minute.
The backbone pGGAselect and this PCR fragment were then digested with PacI and NotI for approximately 30 minutes. Afterward, the fragments were purified from the gel. A ligation was set up according to the protocol, and then a transformation was performed. The resulting colonies after an approximate 12-14 hour incubation period were screened by colony PCR. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and ensure no mutation.

Plasmid piG_10a (BBa_K4604021) is composed of the tet promoter (consisting of the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059)), inducible with doxycycline, bluB, (BBa_K4604005), the gene responsible for AdoCbl production, a riboswitch for AdoCbl (BBa_K4604031), mazF (toxin, BBa_K4604011), and the rrnB terminator (BBa_K4604003). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance.

Cloning Strategy

Plasmid piG_03 (BBa_K4604019) served as the backbone and was amplified according to the Q5 polymerase protocol with an annealing temperature of 56°C and an elongation time of 4 minutes. For the PCR of the insert (bluB), we also used the general protocol for the Q5 polymerase with an annealing temperature of 65°C and an elongation time of 1 minute. Subsequently, a Dpn1 digest was carried out at 37°C for an hour. Afterward, the DNA was loaded onto an agarose gel, and the correct bands were cut out and extracted.
Gibson Assembly was employed according to the protocol to assemble the plasmid. Following transformation, the resulting colonies after an approximate 12-14 hour incubation period were screened by colony PCR. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and ensure no mutation.

Plasmid piG_10b (BBa_K4604022) is composed of the tet promoter (consisting of the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059)), inducible with doxycycline, bluB, (BBa_K4604005), the gene responsible for AdoCbl production, a riboswitch for AdoCbl (BBa_K4604031), the modified T7 RBS (BBa_K4604004), mazF (toxin, BBa_K4604011), and the rrnB terminator (BBa_K4604003). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance. Plasmid piG_10b is identical to piG_10a, with an additional RBS right after the RBS of the riboswitch.

Cloning Strategy

Plasmid piG_10a (BBa_K4604021) served as the template. For the PCR, we followed the general protocol for the Q5 polymerase with an annealing temperature of 61°C and an elongation time of 5 minutes. Subsequently, a Dpn1 digest was conducted at 37°C for 3 hours. The digested DNA was then loaded onto a 1% agarose gel, and the correct bands were cut out and extracted.
Gibson Assembly was employed according to the protocol, with an extended incubation time of 25 minutes. Following the assembly, a transformation was performed, and the resulting colonies after an approximate 12-14 hour incubation period were used for subsequent steps. Since this step involved only the insertion of an additional RBS, screening was not possible. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and ensure no mutation.

Plasmid piG_11 (BBa_K4604041) is composed of the tet promoter (consisting of the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059)), inducible with doxycycline, kanR (BBa_K4604042), a riboswitch for AdoCbl (BBa_K4604031), mazF (toxin, BBa_K4604011), and the rrnB terminator (BBa_K4604003). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning with chloramphenicol resistance.

Cloning Strategy

Plasmid piG_03 (BBa_K4604019) served as the backbone and was amplified following the protocol for the Q5 polymerase, with an annealing temperature of 55°C and an elongation time of 4 minutes. For the PCR of the insert (kanR), we also followed the general Q5 polymerase protocol, using an annealing temperature of 65°C and an elongation time of 1 minute.
A Dpn1 digest was carried out at 37°C for an hour. Subsequently, the digested DNA was loaded onto an agarose gel, and the correct bands were excised and extracted. Gibson Assembly was employed according to the protocol for plasmid assembly. Following assembly, a transformation was conducted, and the resulting colonies after an approximate 12-14 hour incubation period were screened by colony PCR. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and ensure no mutation.

Plasmid piG_23 (BBa_K4604024) consists of the tet-promoter, which includes the tet repressor (BBa_K4604040) and tet operator (BBa_K4229059), inducible with doxycycline. It also includes mazF (BBa_K4604011) and a terminator (BBa_K4604043). The backbone used was pGGAselect, a plasmid commonly employed in Golden Gate cloning that provides chloramphenicol resistance.
PiG_23 contains the tet-repressor without the site-directed mutagenesis we inserted to conform to iGEM parts standards.

Cloning Strategy

A plasmid provided by our principal investigator (PI) served as the backbone and was amplified according to the Q5 polymerase protocol with an annealing temperature of 55°C and an elongation time of 3 minutes. For the PCR of the insert (mazF expression unit), we followed the general Q5 polymerase protocol with an annealing temperature of 58°C and an elongation time of 4 minutes. A DpnI digest was conducted at 37°C for an hour; subsequently, the DNA was loaded onto an agarose gel. The correct bands were excised and extracted. Gibson Assembly was employed according to the protocol for assembling the plasmid. A transformation was performed, and the resulting colonies, after an approximate 12-14 hour incubation time, were screened by colony PCR.
DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to confirm correct insertion and no mutation.

Plasmid piG_23b (BBa_K4604025) is made up of the tet-promoter (consisting of the tet repressor (BBa_K4604040) and tet operator (BBa_K4229059)), that can be induced with doxycycline, the RBS from the AdoCbl riboswitch (BBa_K4604044), mazF (BBa_K4604011), and a terminator (BBa_K4604043.) The backbone we utilized was pGGAselect, a plasmid commonly used with Golden Gate cloning that has a chloramphenicol resistance.

Cloning Strategy

Plasmid piG_23 (BBa_K4604024) was used as a template. For the PCR, we followed the general protocol for the Q5 polymerase with an annealing temperature of 57°C and an elongation time of 3 minutes. A DpnI digest was performed at 37°C overnight. Subsequently, the DNA was loaded onto an agarose gel, and the correct bands were excised and extracted. Gibson Assembly was conducted according to the protocol to assemble the plasmid. A transformation was carried out, and the resulting colonies, after an approximate 12-14h incubation time, were selected for the next steps. Since this involved only a change in the RBS, screening was not feasible. DNA from potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to ensure correct insertion and the absence of mutations.

Plasmid piG_K12BSa (BBa_K4604026) consists of the lacI promoter (BBa_K4604039), the K12 riboswitch from E. coli K12 located in the 5´-UTR of the btuB gene (BBa_K4604031), the lacI repressor gene (BBa_K404703), the inducible trc promoter (BBa_K1950000)—regulated by LacI—and sfGFP with the rrnB terminator (BBa_K4604003). The backbone, designated as pIG23_03, was provided by iGEM Freiburg 2022.

Cloning Strategy

The components, including the lacI promoter, lacI gene, trc promoter, sfGFP, and rrnB terminator, were provided by iGEM Freiburg 2022 on plasmid pIG23_04. We selected plasmid pIG23_03 as our backbone, which included an antibiotic resistance gene (spectinomycin) and Ori. Utilizing Gibson Assembly and AQUA cloning, we integrated sfGFP, trc promoter, lacI, and lacI promoter from pIG23_04 into pIG23_03. PCR was performed on both plasmids to amplify the required parts and add correct overhangs using Gibson primers.
After assembling the two plasmid parts, the resulting plasmid, referred to as the sensor backbone, was used as a PCR template to introduce the correct overhangs for inserting the riboswitch between the lacI promoter and lacI gene.
The sequence for the K12 riboswitch was sourced from E. coli K12, specifically from the 5´-UTR of the btuB gene (National Library of Medicine gene ID: 948468). We obtained the DNA for the K12 riboswitch from IDT and used it as a template for PCR, following a general protocol for the Q5 polymerase with varying parameters (elongation time and annealing temperature):

Your description is quite detailed and follows a clear experimental procedure. I've made some minor adjustments for clarity and flow:
The DNA extracted from plasmid fragments, pIG23_04 and pIG23_03, was loaded onto an agarose gel. After cutting out the correct bands, plasmid fragments of pIG23_03 and pIG23_04 were initially assembled to create the sensor backbone using Gibson Assembly and AQUA cloning, following the respective protocols. Subsequently, a transformation was carried out, and colonies obtained after approximately 12-14 hours of incubation were screened using colony PCR. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to confirm correct insertion and check for mutations.
Using the sensor backbone as a template, PCR was performed to add the correct overhangs for inserting the riboswitch between the lacI promoter and lacI. After a Dpn1 digest at 37°C for an hour, the DNA was loaded onto an agarose gel, and correct bands were cut out and extracted.
PCR for the riboswitch was performed, and the resulting DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. The riboswitch and sensor backbone were assembled using Gibson Assembly and AQUA cloning as per the protocols. Another transformation was carried out, and colonies obtained after an approximate 12-14 hours of incubation were screened by colony PCR. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to verify correct insertion and check for mutations.

Plasmid piG_K12BSb (BBa_K4604027) is made of the lacI promoter (BBa_K4604039), the AdoCbl riboswitch from E. coli K12 from the 5´-UTR of the btuB gene (K12 riboswitch, BBa_K4604031), the ribosome binding site (RBS) from TetR promoter of piG_23 (modified T7 RBS, BBa_K4604004), the lacI repressor gene (BBa_K4047031), the inducible trc promoter (BBa_K1950000), which is repressed by LacI, and sfGFP with a rrnB terminator (BBa_K4604003). The backbone is pIG23_03 which was provided by iGEM Freiburg 2022.

Cloning Strategy

Plasmid piG_K12BSa (BBa_K4604026) served as a template for PCR to insert a stronger RBS from the TetR promoter (modified T7 RBS) directly behind the riboswitch. Gibson primers were designed to have the RBS as their overhang/complementary region. For the PCR, we followed the general protocol for Q5 polymerase with an annealing temperature of 72°C and an elongation time of 6 minutes. A DpnI digest was conducted at 37°C overnight. Subsequently, the DNA was loaded onto an agarose gel. The correct bands were excised and extracted. Gibson Assembly and AQUA cloning were performed according to the protocols to assemble the plasmid. A transformation was carried out.
As the alteration in the plasmid involves only an RBS with a length of merely 14 bp, a colony PCR was not possible as a screening method. The difference in length wouldn't have been noticeable on the gel. Instead, DNA from several colonies was isolated from overnight cultures (5 mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to confirm correct insertion and no mutations.

Plasmid piG_PFBS (BBa_K4604028) is composed of the lacI promoter (BBa_K4604039), the AdoCbl riboswitch from Propionibacterium Freudenreichii located in the 5´-UTR of the cbiB gene [1] (PF riboswitch, BBa_K4604032), the lacI repressor gene (BBa_K404703), the inducible trc promoter (BBa_K1950000), which is repressed by LacI, and sfGFP with an rrnB terminator (BBa_K4604003). The backbone is called pIG23_03 and was provided by iGEM Freiburg 2022.

Cloning Strategy

The lacI promoter, lacI gene, trc promoter, sfGFP, and rrnB terminator were provided by iGEM Freiburg 2022 on a plasmid called pIG23_04. We selected plasmid pIG23_03 as our backbone, which includes an antibiotic resistance (spectinomycin) gene and Ori. SfGFP, the trc promoter, lacI, and the lacI promoter were extracted from pIG23_04 and integrated into pIG23_03 using Gibson Assembly and AQUA cloning. Both plasmids served as templates for PCR to amplify the parts and add correct overhangs via Gibson primers.
After assembling the two plasmid parts, this new plasmid, the sensor backbone, was once again used as a PCR template to incorporate the correct overhangs for the insertion of the AdoCbl PF riboswitch between the lacI promoter and lacI gene. The synthesized DNA of the PF riboswitch from IDT was used as a template for PCR. For this step, we followed the general protocol for Q5 polymerase with varying parameters (elongation time and annealing temperature):

The DNA of the plasmid fragments, pIG23_04 and pIG23_03, was loaded onto an agarose gel. After cutting out and extracting the correct bands, plasmid fragments of pIG23_03 and pIG23_04 were initially assembled to form the sensor backbone using Gibson Assembly and AQUA cloning, following the provided protocols. A transformation was performed, and the resulting colonies were screened by colony PCR after an approximate 12-14h incubation time. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to confirm correct insertion and no mutation.
Next, using the sensor backbone as a template, a PCR was conducted to create overhangs for the insertion of the riboswitch between the lacI promoter and lacI. A Dpn1 digest was carried out at 37°C for an hour, and then the DNA was loaded onto an agarose gel. After cutting out and extracting the correct bands, the PCR for the PF riboswitch was also performed, and the DNA was loaded onto an agarose gel. Following the same procedure, correct bands were cut out and extracted.
The riboswitch and sensor backbone were assembled via Gibson Assembly and AQUA cloning according to the provided protocols. Another transformation was performed, and the resulting colonies, after an approximate 12-14h incubation time, were screened by colony PCR. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to verify correct insertion and no mutation.

Plasmid piG_SYBS (BBa_K4604029) is composed of the lacI promoter (BBa_K4604039), the AdoCbl riboswitch from Salmonella typhimurium/enterica located in the 5´-UTR of the btuB gene [2] (SY riboswitch, BBa_K4604033), the lacI repressor gene (BBa_K404703), the inducible trc promoter (BBa_K1950000), which is repressed by LacI, and sfGFP with a rrnB terminator (BBa_K4604003).

Cloning Strategy

The lacI promoter, lacI gene, trc promoter, sfGFP, and rrnB terminator were provided by iGEM Freiburg 2022 on a plasmid called pIG23_04. We selected plasmid pIG23_03 as our backbone, incorporating an antibiotic resistance (spectinomycin) gene and Ori. SfGFP, the trc promoter, lacI, and the lacI promoter were extracted from pIG23_04 and cloned into pIG23_03 using Gibson Assembly and AQUA cloning. Both plasmids served as templates for PCR to amplify the parts and add correct overhangs via Gibson primers.
After assembling the two plasmid parts, this new plasmid, the sensor backbone, was again used as a PCR template to clone the correct overhangs for the insertion of the AdoCbl SY riboswitch between the lacI promoter and lacI gene. The synthesized DNA of the SY riboswitch from IDT was used as a template for PCR. For this step, we followed the general protocol for Q5 polymerase with varying parameters (elongation time and annealing temperature):

The DNA of the plasmid fragments, pIG23_04 and pIG23_03, was loaded onto an agarose gel. After cutting out and extracting the correct bands, plasmid fragments of pIG23_03 and pIG23_04 were initially assembled to form the sensor backbone using Gibson Assembly and AQUA cloning, following the provided protocols. A transformation was performed, and the resulting colonies were screened by colony PCR after an approximate 12-14h incubation time. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to confirm correct insertion and no mutation.
Next, using the sensor backbone as a template, a PCR was conducted to create overhangs for the insertion of the riboswitch between the lacI promoter and lacI. A Dpn1 digest was carried out at 37°C for an hour, and then the DNA was loaded onto an agarose gel. After cutting out and extracting the correct bands, the PCR for the PF riboswitch was also performed, and the DNA was loaded onto an agarose gel. Following the same procedure, correct bands were cut out and extracted.
The riboswitch and sensor backbone were assembled via Gibson Assembly and AQUA cloning according to the provided protocols. Another transformation was performed, and the resulting colonies, after an approximate 12-14h incubation time, were screened by colony PCR. DNA from potential colonies containing the insert was isolated from overnight cultures (5 mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to verify correct insertion and no mutation.