The ‘Design’ page of the iGEM team at Lanzhou University describes in detail the purpose, working principle and effects of the three components of the engineered probiotic system for IBD designed by our team. On this page, we explain in depth the roles of the diagnostic, therapeutic, and suicide components of the system, how it works as a whole, and how it works. In the overall system, we focus on the selection of chassis organisms, the sensitivity and specificity of the two-component Ths S/R system, the workings of the optimised cytosine base editor TadA-CDd V106W and the design of the bacterial suicide system. In addition to this, this page also describes some proof-of-concept questions about the system we have developed. You can click on the sidebar to quickly jump to the corresponding subheadings for browsing.

Figure 1

Escherichia coli Nissle 1917 (EcN) is an Escherichia coli strain which is serotype O6:K5:H1, with a short LPS side chain, and EcN has a serosensitive K5 type of pods, so it is easily cleared by serum and is not pathogenic. In addition to this, the unmodified wild-type E. coli strain does not produce any enterotoxin or cytotoxin, and thus is harmless to humans and has a good safety profile. EcN can train the immune system, thus preventing the colonisation of the gut by the infectious progenitors of pathogens, such as pathogenic Escherichia coli. It also prevents the colonisation of the gut by pathogens effectively through competitive inhibition.

We chose EcN as a chassis organism not only because it has a safety record in clinical trials for use as an oral drug for the treatment of human gastrointestinal disorders^[1], but also because EcN tends to thrive in the highly oxidative environment of the inflamed intestinal epithelium^[2], and although, similar to many other bacteria in the Enterobacteriaceae family, EcN can be present in the colon but has suboptimal adherence capacity^[3], this deficiency can be be optimised by other means for its colonisation properties. Therefore, we chose EcN to transform and express our designed IBD therapeutic system.

Our system consists of three components, a diagnostic system, a therapeutic system, and a suicide system, which perform the functions of recognising and responding to biomarkers, amplifying signals and releasing drug proteins, and programmed suicide after the patient's symptoms improve respectively.

The targeted release of the parasite-derived drug protein AvCystatin in the patient's intestine is achieved by a two-component self-regulatory system in response to thiosulfate. The release of the drug protein AvCystatin in patients was effectively controlled while amplifying the response regulation signal through a cytosine base editor. The release of the drug protein AvCystatin was terminated in time by the design of a dual chemical substance-regulated switch and a nitric oxide-regulated suicide switch, thus effectively improving the safety of the whole system.

Figure 2. Overall system

Without early diagnosis and treatment, patients with IBD are prone to increased risk of depression, colorectal cancer and even death^[5]. Currently, the clinical means adopted for IBD diagnosis tend to be invasive such as colonoscopy, which is often physically and mentally traumatic and leads to patients' reluctance to frequently follow the progress of the disease^[6,7]. Therefore, non-invasive monitoring of biomarkers of IBD is crucial for IBD patients^[8,9]. In addition, because patients with IBD experience intermittent disease flares, immediate detection and treatment of inflammation can halt disease progression. Moreover, in situ production and delivery of anti-inflammatory drugs can effectively reduce the risks and side effects associated with high-dose systemic drug therapy. Intelligent whole-cell biosensors developed through synthetic biology are a promising noninvasive diagnostic strategy. It has been reported in the literature that some probiotics have been successfully modified into bacterial sensors that can sense disease markers in the gut^[10-14].

Thiosulfate (S₂O₃²⁻) is an attractive target to study the link between intestinal sulfur metabolism and inflammation, Studies have shown that sulfate-reducing bacteria (SRB) present in the colon produce hydrogen sulfide (H₂S) from oxidised sulfur species derived from the host and diet, which is a process that has been suggested to be involved in colitis ^[15] , and that host enzymes subsequently detoxify H₂S detoxification to thiosulfate ^[16-18]. In 2017, Kristina N-M Daeffler et al. screened and reported for the first time by bioinformatics that sha_3128 /9 is the first known thiosulfate sensor (Figure 2 ), Ths S/R, that can be activated by colonic inflammation, with a high level of specificity and sensitivity ( Figure 3 ), suggesting that thiosulfate may be a new biomarker and that the developed bacterial sensor has the potential to be a non-invasive diagnostic for colitis ^[19].

Figure 3. ThsS/ThsR gene locus and domain layout

A. The Shewanella halifaxensis genomic region containing the thiosulfate reductase operon, PhsAB and PsrC (Shal_3125-7), neighboring thiosulfatesensing TCS, ThsS/R (Shal_3128/9), and the ThsR activated promoter (P_phsA). B. Predicted domain architecture of ThsS and ThsR. Residues involved in phosphotransfer are indicated with arrows. ( Kristina N-M Daeffler et al.，2017 )

Figure 4. Characterization of the thiosulfate sensor ThsSR

A. Schematic of ligand-induced signaling through ThsS/R and plasmid design of the aTc- and IPTG-inducible sensor components. B. Ligand response in wild-type and inactivated mutant sensors. White bars are with no thiosulfate and black bars with 5 mM thiosulfate. C. Thiosulfate dose–response curve. D. Selectivity of ThsS/R to thiosulfate over other terminal electron acceptors. All ligands were tested at 10 mM concentration. Data information: Data are mean of at least three biological replicates ± SD. ( Kristina N-M Daeffler et al.，2017 )

Based on the analysis above, in order to construct a genetic circuit for thiosulfate response in EcN, our diagnostic system ( Figure 4 ) used the Ths S/R two-component system reported by Kristina N-M Daeffler et al ^[19] and was constructed in the pSB1C3 plasmid to sense elevated thiosulfate levels in the intestinal tract of the patient to initiate the subsequent therapeutic component.

Figure 5. Diagnostic system

The figure 4 demonstrates our diagnostic system with thsS and thsR driven expression by P_j23104 and P_j23100 promoters, respectively ^[19,22]. Due to the elevated thiosulfate levels in the intestine of IBD patients, constitutively expressed thsS is phosphorylated under the induction of thiosulfate, and the phosphorylated thsS protein can further phosphorylate thsR protein, which leads to the formation of dimers of thsR protein, which in turn drives the expression of subsequent base editing, therapeutic proteins, and so on, through the activation of the thiosulfate-inducible promoter P_phsA.

A well-known application of the CRISPR/Cas9 system is the single-base editor (later some authors developed the double-base editor), which consists of programmable DNA-binding proteins fused with deaminases to precisely change nucleotides at the target genomic locus without double-strand breaks ^[20-21]. Cytosine base editors (CBEs) may have higher off-target activity and lower target base editing activity due to their larger size compared to current adenine base editors (ABEs). Therefore, Monica E. Neugebauer et al. 2022 performed cytidylic acid deoxygenation via phage-assisted sequential evolution to develop a CBE with low off-target activity and high targeted base editing activity similar to the size of existing ABEs ^[23]. They reported that TadA-CD cytosine base editors (TadCBEs) have higher base editing activity at various sites in mammalian cells compared to currently used CBEs, such as BE4max, evoAPOBEC1-BE4max (evoA), and evoFERNY-BE4max (evoFERNY), and exhibit similar or higher C-G to T-A editing efficiency, with lower Cas non-dependent off-target DNA and RNA editing. In addition, the addition of the V106W mutation further reduced off-target editing of TadCBEs, concentrating the editing window at 4-5 base pairs while maintaining higher target C-G to T-A editing and lower A-T to G-C editing ^[23].

Therefore, considering the three bases of ATG in the promoter sequence of the drug protein AvCystatin, we designed to replace it with an ACG sequence with a tag5 tag and chose to employ TadA-CDd V106W ^[23]to control the expression of the drug protein AvCystatin by controlling the P_phsA promoter of the TadA-CDd V106W with the diagnostic system coupling, so that whether or not the diagnostic system detects elevated thiosulfate levels in the patient's intestine determines whether or not the therapeutic system is activated. Based on this, the key to our therapeutic system is TadA-CDd V106W, a CBE optimised to replace the C in the ACG sequence with a tag5 tag in the promoter sequence of the drug protein AvCystatin with a T, which drives the expression of AvCystatin to achieve therapeutic effect.

Figure 6. Therapy system

In our therapeutic system, because the diagnostic system and the entire system of the therapeutic system are constructed on the same plasmid, which may cause many problems such as the vector is too large, which makes it difficult for the expression vector to enter into the chassis organisms through transformation, and even if it is successfully transformed it is still difficult to express all of the target genes, etc., we have constructed the diagnostic system and the components related to the expression of the drug protein AvCystatin in pSB1C3 expression vector, and the Hly BD system, which is related to drug protein secretion, was constructed in another expression vector, pET-9a. Since the P_phsA promoter drives the expression of TadA-CDd V106W under the activation of the dimer formed by the two ths R proteins, TadA-CDd V106W-mediated monobasic editing is induced by the level of thiosulfate. Since the ATG in the promoter sequence of our drug protein was replaced by the ACG-tag5 sequence with higher CBE editing efficiency to silence the expression of target proteins, TadA-CDd V106W expressed proteins can be guided by gRNAs whose expression is driven by the promoter Pj23119 to recognise the ACG-tag5 sequence, and the drug protein is converted to the ACG-tag5 sequence by the principle of CRISPR AvCystatin promoter sequence by replacing the C in ACG with a T, thereby initiating the expression of the parasite-derived drug protein AvCystatin ^[22].

In addition, on a plasmid constructed with pET-9a as the backbone, in order to enable the successful secretion of the drug protein AvCystatin into the extracellular compartment, we integrated the α-hemolysin secretion system[24] into the genetic circuit, which is controlled by the constitutive promoter P_j23104 ^[5]. The drug protein AvCystatin can be released extracellularly with the assistance of the Hly BD secretion system ^[5] to reach the lesion, thus achieving therapeutic effects by increasing or decreasing the levels of certain cytokines.

Nitric oxide is an important inflammatory marker in IBD ^[25], produced by an inducible up-regulated inducible nitric oxide synthase (iNOS) catalysing the production of one molecule of L-arginine to produce one molecule of L-citrulline and one molecule of nitric oxide because of inflammatory response ^[26] , which participates in cell-cell communication in eukaryotic cells, and is a natural inflammation marker ^[25], and thus could serve as an engineered probiotic ideal signals in genetic circuits.

Many bacteria have different nitric oxide sensors, but our selected bacterial enhancer-binding protein NorR reacts only with nitric oxide and not with other reactive nitrogen, with very high specificity ^[28]. In the norR - norV intergenic region, NorR binds to three conserved sites in the norV promoter (PnorV) ^[29,30], and its specific mechanism of action is as follows: in the absence of nitric oxide, the GAF structural domain at the N-terminal end of NorR blocks the binding of the NorR AAA+ structural domain to the bacterial transcription factor, σ54, and thus prevents the transcription of genes; whereas when nitric oxide binds to NorR, the GAF structural domain relaxes repression of the AAA+ structural domain, allowing it to bind σ54 and transcription to occur ^[29,30]. Therefore, we placed the expression of cI deterrent proteins under the control of the PnorV promoter and combined the expression of cI deterrent proteins with a nitric oxide sensor, thus regulating the expression level of cI deterrent proteins through nitric oxide, another marker in the intestinal tract of patients ^[27].

Figure 7. Structural features of NorR ( Matthew Bush et al.,2011 )

A. Domain architecture of the bEBP NorR showing the N-terminal regulatory GAF domain (purple) containing a non-haem iron centre, the central ATPase-active domain (red) and the C-terminal DNA-binding domain (green) that contains an HTH motif. B. Proposed model of the NO-sensing non-haem iron centre in the NorR regulatory domain. C. Theσ54-interaction surface of the AAA+ domain of NorR is the target of GAF-mediated repression.

Figure 8. Model of NorR-dependent activation of norVW ( Matthew Bush et al.,2011 )

During the construction of expression vectors, several phage promoters other than T7 have been of interest to many authors due to their strong transcriptional activity. The first to be developed was the λ phage-derived PL promoter, which is a strong promoter controlling early transcription by the RNA polymerase of E. coli ^[31]. In wild-type λ phage, the transcription of the PL promoter determines the entry of the λ phage into the lytic or lysogenic cycle. The CI gene expression product, which is controlled by the λ phage PE promoter, is a deterrent to the transcription of the PL promoter and is therefore commonly used to regulate the PL promoter. This regulation is mainly divided into two ways: one uses the temperature-sensitive mutant of CI gene, cI857(ts), to regulate the transcription of PL promoter ^[32-33], where the deterrent exists in an active form at a lower temperature (30°C) to deter the transcription of PL promoter, and is deregulated at a higher temperature (42°C) to release its deterrent effect on the PL promoter; and the second regulates PL promoter transcription through the modulation of the CI gene expression products to regulate PL promoter transcription, such as the pLex system (Invitrogen/ThermoFisher), which integrates CI genes rigorously regulated by tryptophan manipulators into chromosomes, and controls the switching of tryptophan manipulators through tryptophan deficiency or not, which in turn regulates the expression of the CI genes, and ultimately, the transcriptional activity of the PL promoter.

Considering that changing the temperature to regulate the transcription of the PL promoter may affect the growth status of the engineered probiotic and that high temperatures may tend to result in reduced solubility of the expressed eukaryotic proteins ^[31]. Therefore, we decided to adopt the second regulation and make appropriate modifications to fit our designed system. We placed the CI gene under the control of the PnorV promoter, thus coupling the nitric oxide sensor component to the expressed portion of the bacterial suicide protein MazF ^[34], and ultimately the signal, the level of nitric oxide in the intestinal tract of the patient, is used to achieve bacterial suicide regulation through this genetic circuit.

Figure 9. Suicide system

In the suicide system we designed, because all the systems we designed were constructed on the same plasmid, the vector would be too large, which would make it difficult to enter the chassis organisms through transformation, and even after successful transformation, it would still be difficult to express all the target genes, etc., we constructed the diagnostic system and the components related to the expression of the drug protein AvCystatin in the pSB1C3 expression vector, while the components related to the secretion of the drug protein AvCystatin in the pSB1C3 expression vector. We constructed the diagnostic system and the components related to the expression of the drug protein AvCystatin in the pSB1C3 expression vector, and all the components of the Hly BD system and the suicide system related to the secretion of the drug protein in the other expression vector, pET-9a.

Figure 10. A suggested model for the inhibition of the recA- and lexA-mediated apoptotic-like pathway by the EDF-mazEF pathway. ( Ariel Erental et al.,2012 )

While the patients gradually recovered under the treatment of drug proteins, the continuous release of drug proteins may lead to the disturbance of the immune system in the patients. Therefore, we designed a suicide system to terminate the release of drug proteins in time. When the patient's IBD symptoms improve, the patient's intestinal nitric oxide level decreases, which reduces binding to the bacterial enhancer-binding protein NorR, which in turn reduces its binding to the bacterial transcription factor σ54, which enhances the inhibitory effect on the PnorV promoter and reduces the expression level of the cI-retarding protein. Subsequently, the reduction in the expression level of the cI deterrent protein relieves its inhibitory effect on the PL promoter, thereby allowing the expression of the MazF protein gene under the control of the PL promoter. The expressed MazF protein triggers programmed bacterial death through cleavage of RNA ^[34] ( Figure 9 ), thereby terminating the release of the AvCystatin drug protein.

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