Synthetic biology-based portable in vitro diagnostic platforms

Early diagnosis of infectious diseases represents powerful means to increase patient survival rate, avoid disease spreading, and decrease healthcare costs. Current Polymerase Chain Reaction (PCR)- and antibody-based diagnostic methods for detecting pathogens offer rapid analysis with highly accurate and specific results. However, those methods are still hampered by the need of sophisticated infrastructures and highly-skilled technicians, which limit the deployment in developing area. Synthetic biology with its rational and short design-to-production cycles has the potential to overcome those limitations. Here, we discuss two promising efforts for pathogen nucleic acids detection using synthetic biology approaches: Synthetic RNA-based and Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated (CRISPR/Cas)-based biosensors. The two systems were reported to show remarkable specificity and sensitivity on detecting and reporting the presence of pathogen via pathogen nucleic acid recognition with lower development and operational costs when compared to current PCR- and antibody-based diagnostic tools. Moreover, both systems can be applied to paper-based platforms which simplify the distribution and utilization in low resource-settings.Keywords: In vitro diagnostics, Synthetic biology, RNA-based biosensors, Toehold switch, CRISPR/Cas syste

Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction

Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction

Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line

Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells

Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line

Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells

Improvement of Plasmid Volumetric Yield by Addition of Glycerol and Phosphate Buffer in Escherichia coli TOP10 Batch Culture

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period

Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

The cholera toxin B pentamer (CtxB5), which belongs to the AB5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB5 and the related heat labile enterotoxin LTB5 have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them

Design of Adenovirus 5 Vector with Adenovirus 26 Hexon Hypervariable Region Sequence using In Silico Approach

Adenovirus type 5 (Ad5) is one of the vaccine vectors, including the COVID-19 vaccine. Pre-existing immunity to Ad5 may suppress the immunogenicity and efficacy of adenovirus vectored vaccine. The neutralizing antibodies are directed specifically toward seven hypervariable regions (HVR) of hexon proteins located on the outer surface of the capsid. This study aims to design an Ad5 vector that may circumvent anti-Ad5 immunity by designing a chimera Ad5 vector with the sequence of Ad26 HVR (Ad5HVR26) using in silico approach. Substitution of the Ad5 HVR DNA sequence may affect the alternative splicing process of adenovirus mRNA, which then influence the protein product. The splice site prediction of Ad5HVR26 chimera vector was found at HVR5, 6, and 7. The codon change in the splice site was performed to decrease the possibility of incorrect splicing, while retaining the original amino acid sequence. The HVR substitution in chimera vector Ad5HVR26 may also affect the interaction of hexon in the capsid. The HVR2 and HVR4 hexon proteins individually interact with other hexon proteins and IX protein. Thus, two designs of the Ad5HVR26 chimera vector were created in this research. The first design was the Ad5 chimera vector with complete substitution of HVR hexon by Ad26 sequence, with codon modification on the splice site. The second design was Ad5HVR26 chimera vector without the HVR2 and HVR4 substitution to maintain the hexon protein interaction with the capsid proteins. Production of the designed vectors are needed to prove the reduction of vector neutralization by pre-existing immunity

Reassembly of CtxB as function of pH.

<p><b>A. Reassembly of CtxB into SDS stable pentamer.</b> Equal amount of reassembled CtxB was applied on each lane of a SDS-PAGE. CtxB reassembled for 0 min and 30 min at pH 7.0 (lanes 2 and 3), or for 30 min at pH 6.0 (lane 4) or and at pH 5.0 (lane 5). Lane 1 is the native CtxB₅. Molecular weight standards are indicated in kDa on the left of the gel. The respective apparent positions of the native CtxB pentamer and of the CtxB monomer are indicated on the right of the gel. <b>B. Reassembly of CtxB into species capable of recognizing GM1.</b> CtxB₅ (○) was treated at pH 1.0 for 15 min and subsequently diluted to a final concentration of 8.6 µM in McIlVaine buffers at indicated pH. The samples were incubated for 30 min at 23°C and analyzed by GM1-Elisa. The results of three independent experiments are shown as a mean ± S.D.</p

Scenari of the steps possibly inhibited by the low pH during the reassembly of CxtB.

<p>Each monomer is represented by a circle. The deprotonated and the protonated CtxB monomers are indicated in white and in black, respectively. In scenari 1, 2 and 3, the β-strands constituting the two subunit interfaces (25–33 a.a. and 97–103 a.a.) are indicated by a line only when they are capable of associating. If the association is impaired by the low pH, the strands of the interfaces are not represented. The native CtxB₅ is represented as a ring of five monomers according to the x-ray crystallographic structure (<i>10</i>). <b>2A. Scenario 1.</b> The folding of the CtxB monomer is inhibited by the low pH. The protonated (black square) and the deprotonated CtxB (white circle) monomers have two different folds, and only the deprotonated CtxB monomer persue the assembly process. The protonated CtxB monomer misfolds irreversibly. <b>2B. CtxB₅ interfaces and histidine residues.</b> For simplicity, out of the five CtxB monomers that composed the native pentamer, only three are shown in strands. Each monomer has two interfaces (Interfaces 1 and 2) involving two different β-strands. The strand number 3 of M (residues 25 to 33) associates with the C-terminal end of the β-strand number 6 of monomer M+1 (residues 97 to 103) to form the interface 1 (I₁). The C-terminal end of the β-strand number 6 of monomer M associates with the strand number 3 of monomer M-1 to form the interface 2 (I₂). The four histidine residues are indicated as balls and sticks, histidines 18 and 94 which are located upstream the two β-strands of the interfaces are colored in black. The figure was made using rasmol and using the coordinates from the x-ray structure of CtxB₅ (<i>10</i>). <b>2C. Scenario 2.</b> The formation of both the interfaces 1 and 2 is inhibited by the low pH. The protonated CtxB monomer is association-incompetent. Only the deprotonated CtxB monomer can associate. <b>2D. Scenario 3.</b> The formation of either interface 1or 2 is inhibited by the low pH. The CtxB protonated can form only one of the two interfaces and is association-deficient. <b>2E. Scenario 4.</b> An intramolecular rearrangement (folding) within the CtxB pentamer is inhibited by the low pH. Both the protonated and the deprotonated CtxB monomers can associate together (black and white) or separately (white-white or black-black). The formation of the native pentamer is pH-dependent. In each scenario, the steps which involve deprotonation/protonation of the CtxB subunit are indicated by a star (*). The native pentamer is considered the most stable species of the reaction and therefore its formation is assumed irreversible.</p

pH-dependence of the association rates determined by SDA (simulation of diffusional Association).

<p>Rates of association were computed at different pH for the wild-type toxin and for the histidine mutants by Brownian Dynamics simulation. <b>A. Rates of association of CtxB subunits.</b> For the sake of clarity only mutants H57N and H18Y-H94N are indicated on the figure. H13N and H13N-H57N gave similar results than H57N and H18Y and H94N gave similar results than H18Y-H94N. <b>B. Rates of association of LTB subunits.</b> The rates of association of wild-type and the two histidine mutants H13N and H57N are shown. The rates of association are calculated for two native independent contacts at an intermolecular distance of 6 Å.</p