Plasmid Design

This plasmid was created for the characterization of our ssDNA optimized mCherry-binding Repebody. It was designed to inducibly express the ssDNA optimized Repebody along with a 6xHis-tag. For this, we used a pET303 bacterial expression backbone. It contains an AmpR gene, encoding for beta-lactamase, which confers resistance to ampicillin. This is used to select transformed bacteria in the lab. The gene of interest is expressed using a T7 promoter and terminator, transcribed by the T7 polymerase. It is regulated by the lactose controlled operon lacO, with the inhibitor protein being expressed by the gene lacI. The backbone also contains a ribosome binding site (RBS), needed for translation of the encoded protein We are expressing this plasmid in the Escherichia coli strain BL21 (DE3), which contains a T7 polymerase also controlled by a lactose operon. This way, the gene of interest is only transcribed after induction of the bacterial culture with Isopropyl-β-D-thiogalactopyranoside (IPTG).

The multiple cloning site (MCS) transcribed by the T7 polymerase contains our ssDNA optimized mCherry-binding Repebody, a short XhoI restriction enzyme scar, and a 6xHis-tag. The XhoI restriction enzyme was used in our cloning strategy to insert the ssDNA optimized Repebody into the vector. The 6xHis-tag is used to detect and purify the ssDNA optimized Repebody in our experiments.

This plasmid was created for the characterization of our ssDNA optimized mCherry-binding Repebody. It was designed to inducibly express the ssDNA optimized Repebody along with a 6xHis-tag. For this, we used a pET303 bacterial expression backbone. It contains an AmpR gene, encoding for beta-lactamase, which confers resistance to ampicillin. This is used to select transformed bacteria in the lab. The gene of interest is expressed using a T7 promoter and terminator, transcribed by the T7 polymerase. It is regulated by the lactose controlled operon lacO, with the inhibitor protein being expressed by the gene lacI. The backbone also contains a ribosome binding site (RBS), needed for translation of the encoded protein We are expressing this plasmid in the Escherichia coli strain BL21 (DE3), which contains a T7 polymerase also controlled by a lactose operon. This way, the gene of interest is only transcribed after induction of the bacterial culture with Isopropyl-β-D-thiogalactopyranoside (IPTG).

The first multiple cloning site (MCS) transcribed by the T7 polymerase contains our ssDNA optimized mCherry-binding Repebody, a short XhoI restriction enzyme scar, and a 6xHis-tag. The XhoI restriction enzyme was used in our cloning strategy to insert the ssDNA optimized Repebody into the vector. The 6xHis-tag is used to detect and purify the ssDNA optimized Repebody in our experiments. The second MCS contains mCherry and an HA-tag. The HA-tag is used to detect mCherry in our experiments.

The gene of interest is expressed using a T7 promoter and terminator, transcribed by the T7 polymerase. It is regulated by the lactose controlled operon lacO, with the inhibitor protein being expressed by the gene lacI. We are expressing this plasmid in the Escherichia coli strain BL21 (DE3), which contains a T7 polymerase also controlled by a lactose operon. This way, the gene of interest is only transcribed after induction of the bacterial culture with Isopropyl-β-D-thiogalactopyranoside (IPTG).

EGFP, standard fluorescent reporter protein. 10xHis affinity tag for detection and purification of recombinant protein. TEV protease site can be used to detach His-tag from EGFP for elution in the affinity column. Used in gel filtrations assays to analyze binding behaviour of the Repebody and for obtaining a standard curve relating fluorescence to protein concentration

mCherry, standard fluorescent reporter protein. 10xHis affinity tag for detection and purification of recombinant protein. TEV protease site can be used to detach His-tag from mCherry for elution in the affinity column. Used in gel filtrations assays to analyze binding behaviour of the Repebody and for obtaining a standard curve relating fluorescence to protein concentration

mTagBFP2, standard fluorescent reporter protein. 10xHis affinity tag for detection and purification of recombinant protein. TEV protease site can be used to detach His-tag from mTagBFP2 for elution in the affinity column. Used in gel filtrations assays to analyze binding behaviour of the Repebody and for obtaining a standard curve relating fluorescence to protein concentration

mCherry, standard fluorescent reporter protein. HA affinity tag for detection, can also be used for purification of recombinant protein. Used in pulldowns and far western blot to analyze binding behaviour of the Repebody.

EGFP, standard fluorescent reporter protein. HA affinity tag for detection, can also be used for purification of recombinant protein. Used in pulldowns and far western blot blot to analyze binding behaviour of the Repebody.

GBP, GFP binding nanobody. 10xHis affinity tag for detection and purification. Sequence includes GGSGG linker between GBP and 10xHis-tag. Used to check expression in E. coli and binding to EGFP (pIG25-111)

mGBP, a mutated version of GBP (F103A, E104R), serves as a non-binding negative control. 10xHis affinity tag for detection and purification. Sequence includes GGSGG linker between mGBP and 10xHis-tag. Used to check expression in E. coli and binding to EGFP (pIG25-111)

Relies on genomic lac-repressor and T7 polymerase of E. coli BL21 (DE3) cells for inducible expression. IPTG inactivates repressor, allowing expression of T7 polymerase, which in turn transcribes gene of interest. Beta-lactamase gene confers resistance to Ampicillin.

6xHis-EGFP, fluorescent reporter protein. 6xHis affinity tag can be used for purification and detection. Was employed in our experiments to test binding with GBP (pIG25-19, pIG25-20, pIG25-24).

Used for inducible expression in E. coli BL21 (DE3) cells. Lac-repressor expressed from lacI gene inhibits transcription by binding lac operon downstream of tac-promotor. IPTG binds and inactivates the repressor, inducing expression. Beta-lactamase gene confers resistance to Ampicillin.

GST as a tag that can be used for detection and purification, thrombin protease site can be used to cleave GST from the rest of the construct. Also contains mTagBFP2 as a standard reporter protein. Was used in our experiments to test binding of (pIG25-19) to EGFP-6xHis (pIG25-17), where it served as a control that EGFP does not binding GST or mTagBFP2.

GST as a tag that can be used for detection and purification, thrombin protease site can be used to cleave GST from the rest of the construct. Also contains mTagBFP2 as a standard reporter protein. GBP is a GFP binding nanobody, this construct was used to evaluate binding to EGFP-6xHis (pIG25-17)

GST as a tag that can be used for detection and purification, thrombin protease site can be used to cleave GST from the rest of the construct. Also contains mTagBFP2 as a standard reporter protein. mGBP is a mutated version of GBP (F103A, E104R) and intended as a negative control, this construct was used to evaluate binding of GBP (pIG25-19) to EGFP-6xHis (pIG25-17).

The pCDFDuet1 backbone is a widely used lac-inducible expression plasmid that encodes two multiple cloning sites (MCS) for expression of two separate coding sequences. Using the CloDF13 origin of replication, it allows us to have a low copy number, and its streptomycin/spectinomycin resistance permits its use in combination with the retron plasmids (pMS366 derivatives), which have a chloramphenicol resistance. For this reason, we decided to use this backbone to insert our gene of interest for retron-mediated mutation.

To achieve an optimal mutation efficiency, we redesigned the vector as follows: One of the coding frames was moved next to the origin of replication (ORI). This is because the integration of ssDNA into a target gene is highly dependent on the direction of DNA replication relative to the DNA’s own reading direction. In order to generate this construct, we deleted the second MCS from pCDFDuet1, a region starting before the second T7 promoter and ending before the plasmid’s only T7 terminator. This region, extended to include the terminator, was then re-inserted between the ORI and the LacI terminator.

The two operons of this plasmid both share a common structure, and are not particularly unique compared to a prototypical operon. Each operon contains a lactose controlled T7 promoter, followed by a ribosome binding site (RBS). Shortly after the RBS, each operon features a different multiple cloning site, in order to allow for selective insertion of new genetic material into each operon. Finally, each operon has a T7 terminator.

This plasmid is a low-copy, temperature sensitive backbone designed to allow retron-based recombineering. Since recombineering is not supposed to take place without regulation, the strict arabinose induction system is included in order to express the retron machinery. The low-copy pSC101 origin of replication is used for this plasmid, since it was determined [1] that this is optimal for expressing the retron for recombineering. This ORI requires the Rep101 protein for copy number control, which denatures at around 37˚C, further drastically reducing the copy number of the plasmid. This plasmid is expressed in bMS.346 E. coli cells, in order to allow for optimal ssDNA and non-coding RNA (ncRNA) expression.

In the development of pMS366 by Schubert et al. [1], the araBAD expression system was chosen in order to express the retron cassette, consisting of the ncRNA, reverse transcriptase and single-strand annealing protein. According to their publication, this was done to enable tight control over the expression of the retron, both in repressing the editing system and in allowing for fine-grained control over its expression when desired [1]. For our purposes, it also allows us to use an additional lac-induced expression system in parallel to this plasmid.

The generation of ssDNA using retrons requires both a reverse transcriptase, as well as an appropriate non-coding RNA. The sequence containing the ncRNA and the reverse transcriptase was originally isolated from the wild-type Eco1 retron, also known as Ec86, from E. coli (Schubert et al. [1]). This ncRNA was modified in order to mutate the cell’s RNA polymerase to be resistant against the antibiotic Rifampicin for the above paper. In order to increase recombineering efficiency, the single-strand annealing protein CspRecT is included, which helps in annealing single-stranded DNA molecules.

This variant of pMS366 was designed for two purposes. Firstly, to demonstrate the capability of the retron system to modify a specific DNA region. Secondly, to investigate the functionality of the RiboJ self-cleaving ribozyme and its effect on the retron ssDNA’s recombination compared to a plasmid without RiboJ. The retron ncRNA in this plasmid is designed to generate a ssDNA fragment that anneals to a known non-coding region close to the ORI. We used this sequence as a primer binding site for our qPCR readout to measure the strength of the retron recombineering system.

In order to test the retron recombineering system against our gene of interest, we engineered a pMS366 derivative containing retron ncRNAs that target the Repebody’s repeat region. This allows us to exchange one repeat variant with another. Here, we introduced two different ncRNA sequences, LRRV targeting retron 1 and LRRV targeting retron 2, each of which causes a mutation in the repeats and thereby generating different Repebody variants. This particular setup is intended to demonstrate a key feature of our approach: the ability to introduce substantial variability into the Repebody by inserting a variable number of ncRNAs. With this we can create a vast library of possible Repebodies.

The first part of the two-hybrid system consists of a Repebody-carrying plasmid. For those we used two slightly different backbones, shown here and on the next slide. The pKNT25 backbone has a wild-type lac promoter and no terminator. Besides the p15A origin of replication, originating from the natural E. coli plasmid p15A and generating a medium number of replications, it carries a neomycin resistance gene (neoR), controlled by an nptII promoter. The resistance gene also works against kanamycin, because both of the antibiotics belong to the aminoglycosides. Therefore, in this instance, their structural differences are negligible, allowing us to use kanamycin as a way to verify successful cloning and as experimental validation in the laboratory. Additionally, the plasmid encodes for the T25 subunit, which is a fragment of the Bordetella pertussis adenylate cyclase toxin (CyaA). As shown in the picture on the left, on this backbone, the MCS is on the N-terminal side of the T25 subunit.

The pKT25 backbone is very similar to pKNT25. The main difference is that the MCS is now on the C-terminal side of the T25 subunit. Both Repebodies (the unmodified mCherry-binding and the ssDNA-optimized mCherry-binding variant) were cloned individually into the multiple cloning sites of the Repebody plasmids. They both have a His-Tag for experimental validation via dot-blot. For each combination, we additionally cloned a plasmid with a GGS-linker between the fusion protein and the T25-subunit. This was done to increase the flexibility of the Repebody, resulting in 8 different constructs. Four constructs with pKNT25 as backbone, and four with pKT25.

The pKNT25 - ssDNA-optimized-mCherry-binding Repebody - 6xHis - GGS plasmid is one of the before-mentioned constructs. Here, it serves as an example to show all the different features. The ssDNA-optimized-mCherry-binding Repebody with a His-Tag, cloned into the backbone on the N-terminal side of the T25 subunit. In between you can see the GGS-linker. This plasmid is one of the constructs used in our selection experiments.

The second half of the two-hybrid system consists of an mCherry-carrying plasmid. The set-up is very similar to how we cloned the Repebody plasmids. Again, we used two slightly different backbones, shown here and on the next slide, with an example plasmid on the third slide. The pUT18 backbone has a wild-type lac promoter and no terminator. It contains the high-copy number origin of replication ColE1 and an ampicillin resistance gene (ampR) controlled by an amp promoter, used to verify cloning and validate our experiments. This plasmid encodes for the T18 subunit, which is the other fragment of CyaA. As shown in the picture on the left, on this backbone, the MCS is on the N-terminal side of the T18 subunit.

As with the Repebody plasmids, the main difference between the pUT18 and pUT18C backbones is the position of the fusion protein relative to the respective subunit. Here, the MCS is on the C-terminal side of T18. We cloned both mCherry and eGFP individually into the multiple cloning sites of the two plasmids. The mCherry is to be bound by the mCherry-binding Repebody, enabling the two CyaA fragments to interact, and the eGFP serves as a negative control. They both have an HA-Tag for experimental validation via dot-blot. For each combination, we additionally cloned a plasmid with a GGS-linker between the fusion protein and the T18-subunit. This was done to increase the flexibility of the fluorescent protein and assess whether it impacts the interaction between the CyaA subunits, resulting in 8 different constructs. Four constructs with pUT18 as backbone, and four with pUT18C.

The pUT18C - mCherry - HA - GGS plasmid is one of the before-mentioned constructs. Here, it serves as an example to show all the different features. mCherry with an HA-Tag, cloned into the backbone on the C-terminal side of the T18 subunit. In between you can see the GGS-linker. This plasmid is one of the constructs used in our selection experiments.

This backbone is a standard for expression of recombinant proteins in mammalian cells. It can be cultivated in bacteria due to its PBR322 origin of replication and ampicillin resistance, isolated and then used for transfection of mammalian cells. For stable expression and propagation, it uses the CMV promoter, bGH_PolyA terminator and the SV40 origin of replication. Additionally, this plasmid can confer the neomycin resistance with the SV40 promoter and SV40 polyA terminator, enabling antibiotic selection for mammalian cells as well.

Fusion construct of three proteins and an affinity tag. Intended for expression in mammalian cells using pcDNA3.1 backbone. Repebody binds mCherry, A1E binds endogenous p62, therefore recruiting any mCherry-labeled target into the lysosomal degradation pathway. Contains mTagBFP2 as a reporter protein for detection via microscopy and FACS, HA affinity tag can be used to perform Co-Immunoprecipitation binding assays checking Repebody and A1E binding and detection via western blot. Sequence includes 4GGS linkers upstream and downstream of mTagBFP2. Used in experiments to target mCherry-labeled proteins for lysosomal degradation.

Fusion construct of two proteins and an affinity tag. Intended for expression in mammalian cells using pcDNA3.1 backbone. Contains mTagBFP2 as a reporter protein for detection via microscopy and FACS, HA affinity tag can be used to perform Co-Immunoprecipitation binding assays checking Repebody binding and detection via western blot. Sequence includes 4GGS linker upstream of mTagBFP2. This construct is supposed to serve as a negative control for degradation regarding pIG25-141 as it lacks A1E, the p62-nanobody which would target a construct for lysosomal degradation.

Fusion construct of three proteins and an affinity tag. Intended for expression in mammalian cells using pcDNA3.1 backbone. GBP binds EGFP, A1E binds endogenous p62, therefore recruiting any EGFP-labeled target into the lysosomal degradation pathway. Contains mTagBFP2 as a reporter protein for detection via microscopy and FACS, HA affinity tag can be used to perform Co-Immunoprecipitation binding assays checking A1E and GBP binding and detection via western blot. Sequence includes 4GGS linkers upstream and downstream of mTagBFP2. Used in experiments to target EGFP-labeled proteins for lysosomal degradation.

Fusion construct of three proteins and an affinity tag. Intended for expression in mammalian cells using pcDNA3.1 backbone. mGBP is a non-binding mutated version of GBP., A1E binds endogenous p62, therefore recruiting this construct toward the lysosomal degradation pathway. Contains mTagBFP2 as a reporter protein for detection via microscopy and FACS, HA affinity tag can be used to perform Co-Immunoprecipitation binding assays checking A1E binding and detection via western blot. Sequence includes 4GGS linkers upstream and downstream of mTagBFP2. Used in experiments as a negative control for targeting EGFP labeled proteins for lysosomal degradation.

Fusion construct of two staple fluorescent proteins, EGFP and mCherry. Intended to be expressed in mammalian cells using the pcDNA3.1 backbone, where the combination of EGFP and mCherry is intended to serve as a readout for ta. EGFP is pH-sensitive, causing its fluorescence to be quenched in the acidic environment of the lysosome. On the contrary, mCherry is unaffected by this environment, which allows the evaluation of lysosomal degradation via the ratio of EGFP fluorescence to mCherry fluorescence. Used in TPD experiments.

Fusion construct of two staple fluorescent proteins, EGFP and mCherry, and FIS1, a sequence causing localization to the outer mitochondrial membrane of mammalian cells. Serves the same function as the previously described EGFP-mCherry regarding its reporter function, but with the addition of FIS1 this construct is intended to be used for evaluation of mitochondrial degradation.

This backbone can be cultivated in bacteria due to its pUC origin of replication and kanamycin resistance, isolated and then used for transfection of mammalian cells. For stable expression and propagation, it uses the CMV promoter. Additionally, this plasmid can confer the neomycin resistance enabling antibiotic selection for mammalian cells as well.

GFP fluorescent reporter protein fused to C-terminus of MTS mitochondria targeting sequence. Intended as a positive control for mCherry-EGFP-FIS1 mitochondria labeling. (pIG25-120).

This backbone can be used for stable expression in mammalian cells, GFP is inserted already, intended to be fused with a protein of interest for fluorescent labeling. It can be cultivated in bacteria due to its pUC origin of replication and kanamycin resistance, isolated and then used for transfection of mammalian cells. For stable expression and propagation, it uses the CMV promoter and the SV40 origin of replication. Additionally, this plasmid can confer the neomycin resistance enabling antibiotic selection for mammalian cells as well.

Contains mTagBFP2 as a fluorescent reporter protein instead of EGFP, plasmid is equally designed for upstream insertion of coding sequence to generate fusion construct. Used in our experiments as a control for cytosolic accumulation of mTagBFP2.

Contains mCherry as a fluorescent reporter protein instead of EGFP, plasmid is equally designed for upstream insertion of coding sequence to generate fusion construct. Used in our experiments as a control for cytosolic accumulation of mCherry.

This backbone has pBR322 origin of replication and ampicillin resistance for propagation in bacteria, from which it can be isolated and then used for transfection of mammalian cells. It uses the CMV promoter and the SV40 origin of replication in the mammalian cell. Additionally, this plasmid can confer hygromycin resistance enabling antibiotic selection for mammalian cells as well.

Codes for fusion construct of wild-type p62 and mCherry. This causes fluorescent labeling of p62, a mammalian protein involved in lysosomal degradation. Used in our experiments as a control for lysosome formation.