The plasmid pEDIT1 is composed of three fragments assembled by Golden Gate cloning using the enzyme BsaI and T4 DNA ligase. Each fragment was previously obtained by PCR from a template plasmid using appropriate primers. These primers have 5' overhangs with BsaI sites possessing suitable protruding ends to allow directional assembly during Golden Gate cloning. Fragment (1) was amplified from the plasmid pENTR puroR R4-R3 using primers Pi28 and Pi29. This fragment (1) carries the attR3-attR4 recombination sites, the kanamycin resistance gene, and the pUC origin of replication (Figure 9). Fragment (2) carrying the chloramphenicol resistance gene and the oriT transfer origin of plasmid RP4 was amplified from plasmid pH2-Cm using primers Pi24 and Pi25. Fragment (3) carrying the ori2 replication origin of plasmid F and the gene encoding the associated replication protein RepE was also amplified from plasmid pH2-Cm using primers Pi26 and Pi27 (see Material page). The primer sequences are provided in the appendix (Table xx)

The different fragments were assembled through Golden Gate cloning (Figure 3), and the ligation mixture was introduced into the DH5a strain by transformation. The transformants were selected on solid LB medium containing the antibiotics chloramphenicol (Cm) and kanamycin (Kan). Four randomly selected clones were characterized. The corresponding plasmids were digested with the restriction enzyme EcoRI and analyzed by agarose gel electrophoresis.

According to the expected map of the plasmid pEDIT1 (Figure 3), there is a unique EcoRI restriction site within the chloramphenicol resistance gene. Correct plasmids should be linear at a size of 5809 base pairs after EcoRI digestion. 2 clones were obtained with the right size, see Results page. The plasmid corresponding to clone 1 was validated by sequencing. This pEDIT1 plasmid has two replication origins, ori pUC, which allows high-copy replication (100 to 500 copies/cell), facilitating the extraction and purification of the pEDIT1 plasmid, and ori2, which allows limited replication (1 copy/cell). This plasmid carries the attR3 and attR4 recombination sites. During Gateway assembly through recombination between attR and attL sites, the ori pUC will be eliminated along with the kanamycin resistance gene, and the final plasmid will correspond to an unstable mobilizable mini F plasmid due to the absence of partition genes, with low copy number, carrying the CmR gene.

In this version, we've added a ccdB sequence which is recommended in a gateway destination vector. We decided to make a golden gate assembly of 5 different fragments.

The first 2 PCR fragments will be the same as those used to build version 1 of pEDIT, Fragment (1) carrying a gene responsible for chloramphenicol resistance and the origin of transfer oriT of the RP4 plasmid; Fragment (2) carrying the replication origin ori2 of the MiniF plasmid and the gene repE coding for the associated replication protein. Fragment (3) carrying the attR4 recombination site and the ccdB cassette; Fragment (4) carrying the origin of replication pucOri and a RifR selection pressure; Fragment (5) carrying the attR3 recombination site.

The construction of plasmid pEDIT2 required obtaining the intermediate plasmid pEDIT2-Cm. This intermediate plasmid is constructed from the two initial plasmids pEN-dCas9-CDA1-UG1 and pdCas9 sold by the company Addgene. The first plasmid carries the base editor dCas9-CDA1-UGI expressed from the recA promoter. Our goal is to replace this recA promoter with the inducible ptet promoter by Atc while introducing the tetR gene encoding the TetR repressor present on the second plasmid pdCas9. To achieve this, the two initial plasmids were digested with the enzymes BamHI and SpeI, followed by ligation with T4 DNA ligase (Figure 5).

The ligation products were introduced into the DH5a strain. Transformants were selected on solid LB medium containing the antibiotics Cm and Kan. Four randomly selected clones were characterized. The corresponding plasmids were digested with the EcoRI enzyme and analyzed by agarose gel electrophoresis. There are three EcoRI restriction sites within the pEDIT2-Cm plasmid, so correct plasmids should generate 3 fragments of respective sizes 4243 bp, 4048 bp, and 1372 bp. All obtained clones had the expected digestion profiles, considering that the two fragments of 4243 bp and 4048 bp co-migrate in the agarose gel (see Results page). In this intermediate plasmid pEDIT2-Cm, we deleted the region responsible for chloramphenicol resistance by double digestion with the enzymes ZraI and MlsI, which create blunt ends. After thermal inactivation of the restriction enzymes at 60°C for 20 minutes and ligation using T4 DNA ligase, the products were introduced into the DH5a strain. Transformants were selected on solid LB Kan medium. After overnight growth at 37°C, a replica was made using a velvet pad (see protocols below) on solid LB Cm medium to identify colonies that had lost chloramphenicol resistance. One of the chloramphenicol-sensitive clones was characterized. The corresponding plasmid was digested with the enzymes SpeI and KpnI and then analyzed by agarose gel electrophoresis. Two fragments of respective sizes 6242 bp and 2811 bp were obtained, which is consistent with the expected digestion profile.

The pEDIT3-gRNAs plasmids correspond to the only variable module of the editing system. These plasmids contain the expression cassettes for specific gRNAs and the attL1 and attL2 recombination sites. Version pEDIT3a contains two gRNAs in tandem expressed from the ptet promoter, whereas vector pEDIT3b contains four gRNAs in tandem. These plasmids require the construction of several intermediate plasmids:

The pHost-spacer plasmids contain the ptet promoter, a ccdB suicide cassette, and the gRNA scaffold necessary for interaction with the Cas9 protein. Each of these plasmids will accept a specific spacer. These plasmids differ in the presence of distinct BbsI sites that allow directional assembly of gRNAs into the recipient vectors pV2-mScarlet or pV4-mScarlet. The pHost-spacer plasmids consist of two fragments obtained by PCR from the template plasmids pINS-Rif and pT1, using primers with 5' overhangs carrying distinct BbsI sites (Figure 6).

Each pHost-spacer plasmid requires two pairs of specific primers (see Material page). The PCR products were purified and then digested with BbsI. After thermal inactivation of the BbsI enzyme at 60°C and ligation with T4 ligase, the products were introduced into the DB3.1 strain resistant to the CcdB toxin. Transformants were selected on solid LB Kanamycin medium. Two independent clones for each different pHost-spacer plasmid were analyzed by BbsI digestion (see Results page). All of them generate two fragments of 1815 bp and 1450 bp.

The expression plasmids pEx-gRNA1 and pEx-gRNA2, containing the spacers S1-oxa48 and S2-oxa48, targeting the Trp25 and Arg163 residues of the carbapenemase Oxa-48, respectively, were obtained through Golden Gate cloning using BsaI and T4 DNA ligase into the plasmids pHost-spacer1 and pHost-spacer2, respectively (Figure 8). This cloning involves prior hybridization of single-stranded oligonucleotides (Pi20 with Pi21 for spacer S1-oxa48 and Pi22 with Pi23 for spacer S2-oxa48) to form double-stranded spacers. The protocol for hybridizing complementary oligonucleotides is described below.

During Golden Gate cloning, the BsaI sites as well as the ccdB suicide cassette are lost, and the spacer is fused with the gRNA scaffold to form a functional gRNA expressed under the control of the ptet promoter, with a transcription terminator located at the end of the gRNA. The Golden Gate cloning products were introduced into the DH5 strain sensitive to the CcdB toxin, allowing elimination of the pHost-spacer plasmids. Transformants were selected on solid LB Kanamycin medium. Independent clones for each version of the pEx-gRNA plasmid were digested with the ZraI enzyme and generate a single 2023 bp fragment.

The plasmids pV2-mScarlet and pV4-mScarlet, which host tandem gRNAs, are derivatives of the plasmid pENTR4-dual sold by Thermofisher, carrying the attL1 and attL2 recombination sites. The goal is to replace the chloramphenicol resistance gene and the ccdB suicide gene located between attL1 and attL2 sites with the gene encoding mScarlet itself flanked by two BbsI restriction sites. These BbsI sites will enable directional insertion of tandem gRNAs through Golden Gate cloning. The construction of these plasmids involves a series of steps outlined in Figure 9. The first step involves eliminating an undesirable BbsI site in the pENTR4-dual plasmid. This was achieved through deletion using the blunt-end enzymes ZraI and MbiI to obtain the plasmid pENTR4∆BbsI. The second step involves amplifying the mScarlet gene from the template plasmid pMCF-mScarlet using primers that introduce XhoI and KpnI sites at the ends, as well as distinct internal BbsI sites. Finally, the last step involves cloning the mScarlet PCR fragment into the pENTR4∆BbsI plasmid through double digestion with XhoI and KpnI.

The ligation products were introduced into the DH5 strain sensitive to the CcdB toxin, allowing the elimination of the pENTR4∆BbsI plasmids. Transformants were selected on solid LB Kanamycin medium. The appearance of pink colonies indicates the presence of mScarlet. Plasmids from these pink clones were digested with BbsI. All of them generated two fragments of 2151 bp and 1014 bp, in line with expectations (see Results page).

As mentioned on the engineering page, in order to improve the selection of the plasmid that will be constructed from pV2-mScarlet and pV4-mScarlet, we have decided to reconstruct this plasmid with streptomycin resistance instead of kanamycin. To do this, we decided to perform a golden gate BsaI assembly between two fragments generated by amplifying two plasmids. The first one is the amplification product of plasmids pV2-KanR-mScarlet and pV4-KanR-mScarlet using primers Pi62 and Pi63 (Figure 10 A). The second fragment is the amplification product of plasmid 206 using primers Pi64 and Pi65 (Figure 10 B).

The PCR products were purified and then digested with BsaI, After thermal inactivation of the BsaI enzyme at 60°C and ligation with T4 ligase, the products were introduced into the DB3.1 strain resistant to the CcdB toxin. Transformants were selected on solid LB streptomycin.

The construction of the pEDIT3a-oxa48 plasmid is carried out through Golden Gate cloning using BbsI from the previously obtained plasmids pEx-gRNA1, pEx-gRNA2, and pV2-mScarlet. The depicted BbsI sites enable simultaneous and directional assembly of the different gRNAs in tandem (Figure 10).

The products of the Golden Gate reaction were introduced into DH5a cells. Transformants were selected on solid LB Kanamycin medium. Eight white clones not expressing mScarlet were characterized. The corresponding plasmids were amplified by PCR using primers Pi20 and Pi23, which hybridize with gRNA1 and gRNA2, respectively. The PCR products were analyzed by electrophoresis on a 1.3% agarose gel. If the tandem construction is correct, we expect a PCR product of 243 bp. Only clones 3 and 7 generated the correct PCR product.

Plasmid pEDIT4 is the final plasmid enabling the construction of plasmid pEDIT5. This contains the sequence of interest between the attR2 and attL3 recombination sites. Initially, pending the development of the Protacs system, we used the pEN-SacB plasmid containing the SacB sequence between the attR2 and attL3 sites for the construction of pEDIT5. However, we have also prepared the plasmid into which the Protacs system will be inserted in order to integrate it into the final pEDIT5 plasmid.

This plasmid, which will be referred to as pEDIT4-empty, is constructed from the plasmid pEN-SacB. Amplification of pEN-SacB is performed with primers Pi36 & Pi37 to amplify the region containing attL3-KanR-Ori-attR2. The PCR product is checked on an agarose gel, and a 2.7 kb fragment is expected. The PCR product is then purified by PCR clean-up and digested with the SpeI enzyme for 1 hour at 37°C. After deactivating the enzyme by heating at 60°C for 20 minutes, Hi-T4 ligation is performed.

For the BacPROTACs, we designed a complex that could degrade OXA48. With a protein specific nanobody on one side, and ClpX, which is part of the bacteria’s system of degradation on the other. To learn more about how we made it and what led us to this final solution, it is possible to visit the Docking page. To test our nanobody, we needed to overproduce the oxa48 protein, so we could run interaction tests.

First of all, to keep the process safe, we used an OXA48 DNA sequence that didn’t coded for the addressing signal, so once produced, the protein couldn’t be addressed to the periplasm and wouldn’t be able to confer the resistance to carbapenems: we amplified it from plasmid pOXA48 DNA. We then added it in the pet28a plasmid (by ligation).Therefore, the construction results in an inactive form of OXA48 and confers no resistance to ß-lactam. First, we runed a test to verify this hypothesis:

We also tested the best conditions for overproduction. When we were sure to have the good plasmid (extraction, digestion and sequence verification), we transformed it into BL21/pLysE strain to overproduce the inactive form of OXA48 which is tagged with 6-his for affinity purification using resin Ni-NTA columns. In the resulting pet28-oxa48 plasmid the oxa gene is under the control of promoter T7 and will be expressed after inducing T7 RNA polymerase by ITPG. Next, we tried to find the good conditions of overproduction, so liquid cultures were started with a large volume of TB medium(richer than LB medium) and then these cultures were subdivided into several tubes containing different quantities of L-rhamnose (a n inducer of lysozyme LysY, an inhibitor of RNA pol T7) and a fixed concentration of ITPG 100µM for RNA Pol T7 induction. After checking the OD, protein extractions were carried out by sonication followed by affinity purification using Ni-NTA columns in order to determine the optimum temperature, time of induction with IPTG and L-rhamnose concentration conditions for overproduction. We ran 11 conditions tests, extracted the protein by sonication and then collected the purified proteins into 7 different fractions after they passed through an Ni-NTA column purification. Overproduction and purification of Oxa48 was analyzed by SDS-PAGE.

Condition	L-rhamnose quantity (µL)	Incubation temperature (°C)	Incubation time (h)
1	2	37	5
2	10	37	5
3	10	37	3
4	10	37	1
5	10	30	3
6	20	37	5
7	10	30	2
8	15	37	5
9	2	37	5
10	10	37	2
11	10	30	1

For each condition, we made a SDS-page gel deposing the 7 fraction we collected through the column. As we eluted with imidazole (that has a structural analogy with the polyhistidine tail and will replace the protein in the interaction with the gel.) And as we didn’t do a purification to take off the imidazole and since it’s charged, it will affect the migration on the sds gel, so the proteins won’t necessarily be in phase with the ladder. We were waiting for a band at 28,15 kDa, and found two conditions that presented a band around that weight : conditions 9 and 3.

After designing and creating our plasmids, it is important to test the efficiency of the Super BugBuster system. To do so, we selected various tests that should be done, however, because of schedule constraints, these tests are still in the making and we need to remain patient before being able to observe the results of our work.

pET28a is the most popular expression plasmid on the market (described in >40,000 published articles). It contains the T7 promoter and an adjacent lac operator sequence that is included to suppress uninduced expression. Translation initiation is mediated by a Shine–Dalgarno (SD) sequence originating from the major capsid protein of T7 (gene 10 protein). In a typical experiment, the coding sequence to be expressed is cloned downstream of, and in frame with, the coding sequence for a poly-histidine purification tag (His6) and a thrombin protease recognition site (TPS) so that the recombinant protein produced can be easily purified using standardized protocols. We just added the sequence we wanted to the plasmid, that already has all the overproduction material. To do so, we did an enzymatic digestion and a ligation with the T4 ligase.

After designing and creating our plasmids, it is important to test the efficiency of the Super BugBuster system. To do so, we selected various tests that should be done, however, because of schedule constraints, these tests are still in the making and we need to remain patient before being able to observe the results of our work.

It is really important to test the sensitivity of the strains we use during experiments against antibiotics, especially if those differential antibiotic resistances are used to select a specific type of bacteria. Therefore, before conjugation, the donor bacteria (MFDpir) carrying pEDIT5 (plasmid containing the Super BugBuster system) and the recipient bacteria (TOP10/pOxa48) should be inoculated on media containing either ampicillin or chloramphenicol. The donor bacteria should be able to grow on a medium with chloramphenicol but not on a medium with ampicillin, while the recipient strain should be able to grow on a medium containing ampicillin and not on a medium containing chloramphenicol.

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. To see if our tool works, we need to test if the plasmid conjugates itself well with other bacteria. Recipient bacteria are selected by transferring recipient and donor bacteria after conjugation to a media containing both ampicillin (resistance found in pOxa48) and chloramphenicol (resistance found in pEDIT5) ( Description page). Conjugation efficiency can be expressed as the ratio between the number of recipient bacteria that have received the conjugative plasmid (bacteria carrying both pEDIT5 and pOxa48) and the total number of recipient bacteria. Formula used to calculate the conjugation efficiency :

After conjugation, a colony from the isolated recipient bacteria should be used to conduct a PCR with primers specific to pEDIT5. This would allow us to verify if the recipient bacteria really received the pEDIT5 plasmid that contains our tool.

A viability test should be done to study the effects of the Super BugBuster system on the growth and survival of the bacteria that are carrying it. A first test should be conducted using the recipient bacteria (TOP10/pOxa48) carrying the plasmid pEDIT5 ( Description page) or not. The two bacterial cultures should be incubated at 37°C in rich liquid media containing ampicillin. After a few hours of incubation, optical density (DO) should be measured and compared between bacteria carrying our tool or not, to characterize the effects of the presence of the pEDIT5 plasmid on the growth and survival of the recipient bacteria. During this test, PCR should be done targeting pEDIT5 to verify if the recipient strain hasn’t lost it since there is no chloramphenicol in the medium. A second test should be conducted using recipient bacteria (TOP10/pOxa48) carrying the plasmid pEDIT5. These bacteria should be incubated in rich liquid media with chloramphenicol at 37°C, with or without anhydrotetracycline. Anhydrotetracycline allows the induction of our tool and the expression of the genes regulated by the tetR/tetA inducible promoter ( Parts page). After a few hours of incubation, optical density (DO) should be measured and compared between bacteria with a tool induced or not, to characterize the effects of the induction of the tool on the growth and survival of the recipient bacteria. During this test, PCR should be done targeting pOxa48 to verify if the recipient strain hasn’t lost it since there is no ampicillin in the medium. The results of these two tests should help us determine if our tool represents a metabolic burden for the recipient bacteria. If, when induced, our tool prevents or slows the growth of the recipient bacteria, we will have to adapt the strategy used to test the efficiency of resensitization.Also, these results should help us find the optimal induction duration and inducer concentration that are the most suited to the use of our tool.

An induction is characterized by an increase in the expression of genes under the regulation of an inducible promoter when in the presence of an inducer. In our case, the inducible promoter tetR/tetA is constitutively repressed and that repression is lifted in the presence of anhydrotetracycline, the inducer (Safety page). After conjugation and transfer of our tool (pEDIT5) to the carbapenem resistant bacteria (TOP10/pOxa48), transferring the recipient bacteria on a media containing anhydrotetracycline should activate the Super BugBuster system and resensitize these bacteria to carbapenems. The efficiency of our tool in resensitizing the targeted antibiotic resistant bacteria would then need to be tested. In order to test the efficiency of the Super BugBuster system in resensitizing carbapenem resistant bacteria, TOP10/pOxa48 bacteria that received pEDIT5 should be transferred from a medium containing ampicillin and chloramphenicol (without DAP, this medium contains only recipient bacteria), to a medium containing anhydrotetracycline, ampicillin and chloramphenicol before being incubated in optimal growth conditions. The number of colonies observed on this medium will represent the “CFU still resistant despite the induction of the Super BugBuster system” (CFU = Colony Forming Units), and it should close to 0 since our tool is supposed to resensitize TOP10/pOxa48 bacteria to ampicillin (a kind of beta lactam like carbapenems). In parallel, TOP10/pOxa48 recipient bacteria should also be transferred on a medium containing anhydrotetracycline and chloramphenicol before being incubated in optimal growth conditions. The number of colonies obtained on this media will represent the “total number of CFU”. The efficiency of resensitization performed by our tool will be calculated thanks to this formula :

The step after the docking work is the experimental testing. To do so, we need first to overproduce and purify our Oxa48 protein and the nanobodies we selected. For that, we need to find the right conditions of overproduction. After the obtention of both proteins purificated, we will need to verify them. First, we will need to do a SDS-page gel to verify protein purity. Then, the homogeneity of the protein (whether its distribution is fine or if there are a lot of aggregates) should be tested, with a dynamic light scattering ( powerful tool for studying diffusion behaviour of macromolecules in solution) combined to a size exclusion chromatography ( separates molecules based on their size by filtration through a gel). Lastly, it is important to verify the protein, by identifying it thanks to a mass spectroscopy test.

Interaction tests are pretty long manipulations that characterize how two proteins react to each other. We principally thought about two tests: a fluorescence test and a NMR one.

• Fluorescence test: The bimolecular fluorescence complementation (BiFC) test has been used to visualize interactions among multiple proteins or position in cells in many cell types and organisms. The BiFC assay is based on the association between two non-fluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragment. It allows direct visualization of proteins interaction and is pretty easy to use. The interaction can therefore be detected without exogenous fluorogenic or chromogenic agents, avoiding potential perturbation of the cells by these agents. This also avoids potential problems caused by uneven distributions of the chromogenic or fluorogenic substrates or ligands. But there is always a delay between the time when the fusion proteins interact with each other and the time when the complex becomes fluorescent. In addition, formation of some bimolecular fluorescent complexes is irreversible at least in vitro.
• NMR test: NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, interaction, dynamics and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Another advantage of this test is that it can be performed in a lot of different conditions and states: solid, solution, membranous environment. The common procedure of protein NMR structural determination usually includes four stages: (1) isotope-labeled protein sample preparation, (2) NMR data collection and analysis, particularly assigning the chemical shifts of the 1H, 15N, and 13C atoms in the protein molecule, (3) structural calculation and refinement using distance and/or orientation restraints derived from specific NMR experiments, and (4) structural quality assessment, each thoroughly introduced and reviewed.NMR analysis of proteins is much less dependent on sample conditions compared to X-ray or cryo-EM methods and is highly sensitive to subtle changes in chemical environments .

As we didn't have much time, we also thought about doing an ELISA test to see if the interaction worked well.

• ELISA is a laboratory technique that detects certain antibodies, antigens and other substances in your blood, pee or other bodily fluid. Laboratory scientists use this technique for several medical tests — from diagnosing infections to confirming pregnancy. It can be used to detect and count certain antibodies, antigens, proteins and hormones in bodily fluid samples. Simpler and quicker than the above.

Many ELISA tests have a positive or negative result, but some might be invalid:

• Positive result: A positive result means that the test detected the substance it was checking for.
• Negative result: A negative result means that the test didn’t detect any of the substance it was checking for.
• Invalid result: An invalid result means there was an error in the testing. This could mean an issue with the sample collection or the test itself. You’ll need to take another test.

In the final step, the laboratory scientist adds a substance that reacts with the enzyme. This makes the substances change color if the antibodies are present. In other words, if the test is positive, then a color reaction will occur. If you don’t have antibodies to that certain antigen (the HIV virus, in this case), there will be no color change.

The degradation test is there to see if our plasmid is well degraded in its environment.

Finally, the last test that we planned to do is the test of functionality between ClpX and the linker Nanoboday, to see if our BacProtac solution is viable, and possible.