Team:Vilnius-Lithuania/Engineering
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overview
As a team, participating in a synthetic biology competition, we heavily relied on the good engineering and scientific practices. However, since synthetic biology is not as well established a field as civil or aerospace engineering is, not only did we employ a so-called “middle-out” approach, but we also tailored the engineering design cycle to our own unique needs. Throughout the project we practiced accurate measurement of our progress wherever possible - from promoter activity to material integrity - carefully documenting our results.
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Detection
1st iteration
HDA
1st iteration
research & imagine
After we discovered the need to empower farmers with the ability to swiftly detect an exact pathogen species, we set a goal to create a rapid and specific detection kit. Having these two properties in mind, we decided to base our tool on isothermal DNA amplification - HDA and lateral flow assay - LFA to visualize the results.
HDA is an easy to use method meant to amplify short DNA sequences, while LFA, based on DNA-DNA hybridization, is a fast way to identify amplified sequences specific to a single pathogen species1. We chose HDA because this amplification does not require expensive lab equipment or scientific background and due to this can be performed in rural farms.
The HDA reaction imitates a natural replication process where helicase is used to unwind DNA and allows amplification to occur at a stable temperature without the need for expensive thermocyclers. This method is also easy to design since it requires only two primers compared to other isothermal amplification methods such as LAMP2.
design
Our team designed this amplification in a way that the reaction is asymmetric, meaning that one primer is used in an excess concentration. This type of amplification generates ssDNA fragments which can be immediately used for our lateral flow assay tests to identify which species of Flavobacterium are present.
build & test
We performed HDA reaction according to the manufacturer's protocol. But we were not satisfied with the results since most of the time no or very little amplicon was seen in an electrophoresis gel. In order to have a good working reaction mix, we needed to optimize reaction conditions.
learn & improve
A fragment from F. psychrophilum genomic DNA was amplified via HDA without any modifications. However, F. columnare proved to be challenging to amplify, so we decided to try a few optimizations approaches:
- Different pairs of primers;
- Optimization of reaction temperature;
- The quantity of gDNA/primers/MgSO4/NaCl/enzyme;
- Two-step HDA reaction;
- Genomic DNA restriction;
- Additives (DMSO, PEG4000, sorbitol, dextran, ficoll, betaine, formamide, pyrophosphatase).
2nd iteration
2nd iteration
build & test
We performed amplification reactions using our chosen optimization methods. Based on the results seen in agarose gel, the most efficient additives were DMSO and sorbitol. The reason why these additives improved HDA assay, is their ability to minimise secondary structures of DNA which improves primer annealing3. However, some of the results did not repeat through a series of experiments, so further optimization should be performed to find the best HDA reaction conditions.
learn & improve
Since no optimization was efficient enough to enhance the reaction, we concluded that primers used for F. columnare gene fragment amplification were designed using wrong parameters. To combat this issue, we decided to redesign primers. Newly created primers were longer and had a greater GC% content, thus increasing their melting temperature. All parameters matched perfectly with the suggested parameters for HDA primers design. After testing those primers, we saw that the reaction efficiency was still too low. Eventually, we concluded that a more sensitive method then electrophoresis should be used to determine the concentration of amplicons such as qubit fluorometer or electrophoresis with a more sensitive dye than ethidium bromide.
1st iteration
LFA
1st iteration
research & imagine
After researching ways to create a point-of-use test, we decided to base our detection tool on lateral flow assay method4. The main reasons for this decision were that it requires no prior training or scientific knowledge to use and is rapid as well as robust. Thus we imagined that it would be the perfect tool for on-site detection. Keeping this in mind, our detection tool will help to monitor bacterial infections in aquaculture farms as well as serve as a foundation for creating a quantitative LFA test to be used not only in farms but in the wild environment as well.
design
LFA test is a universal method and with the utilization of ssDNA probes it can be designed to detect all kinds of DNA amplicons. It must not be forgotten, however, that a high level of specificity has to be achieved for the test to function properly and to get as little false-positive or false-negative results, as possible.
To assure specificity we found marker genes for F. columnare and F. psychrophilum, which were 16S ribosomal RNA genes, and created detection (BBa_K3416101, BBa_K3416107), capture (BBa_K3416102, BBa_K3416108) and control (BBa_K3416103, BBa_K3416109) probes for fragments of those marker genes5.
build & test
Our team analyzed research papers and wrote all protocols as well as designed experiments necessary to create and test a lateral flow assay detection tool. For LFA to work, gold nanoparticles conjugated to the ssDNA detection probes are crucial. To achieve this conjugation, functionalization reaction must be performed. But we ran into a problem and were unable to functionalize gold nanoparticles with ssDNA detection probes using NaCl “aging” method6. After functionalization reaction gold nanoparticles turned deep blue or colorless. These color changes identify that nanoparticles aggregated during the process.
learn & improve
By trying to make functionalization successful, we put a lot of efforts into improving our protocol like using a greater concentration of ssDNA detection probes. Nonetheless, it did not work, and no functionalization occurred. After these unsuccessful experiments, we talked with prof. A. Ramanavičienė and learned about different methodologies for functionalization.
build & test
One of the most promising methods, the professor suggested to us, is based on a low pH environment and is suitable for probes which have a poly-A sequence7. In pH 3 adenine becomes protonated and two poly-A sequences can make parallel duplexes8. Such structures conjugate to gold nanoparticles more efficiently. Since our team created probes with poly-A sequences we thought this method was worth trying. Besides that, we also decided to omit the sulfhydryl group activation and DTT extraction steps, because they can cause unwanted gold nanoparticles aggregation.
learn & improve
After testing, we saw that the functionalization reaction using a low pH method was successful because gold nanoparticles remained red and they did not aggregate during NaCl test so we carried on with our experiments. Shortly after we ran into a second problem and saw that the test created for F. psychrophilum was non-specific and became positive with F. columnare as well as E. coli DNA samples.
2nd iteration
2nd iteration
research & imagine
If the test is non-specific then it can lead to false positive results which would make our test not suitable for F. psychrophilum identification. We needed to find a solution to improve specificity. Our team read various research papers and came to a conclusion that running buffers can affect the performance of the test9. However, if different optimizations with running buffers would not improve specificity a new marker gene for F. psychrophilum identification was found - rpoC gene (JX657167)10.
design
Two strategies were designed. First of all, we wanted to see if buffers with SDS, SSC or formamide which are often used in lateral flow assay tests based on nucleic acid hybridization could improve specificity. Second of all, new ssDNA probes were created for the chosen rpoC gene (BBa_K3416110, BBa_K3416111, BBa_K3416112). This time ssDNA probes had a greater GC% content and were longer.
build & test
To check the first strategy we tested our lateral flow assay test strips using ultrapure water as well as phosphate buffer. Also two running buffers with different SDS and SSC concentrations and one buffer containing formamide were chosen and tested. In addition, new LFA test strips using ssDNA probes created for rpoC gene were developed and tested with DNA amplicons from different bacteria species.
learn & improve
Results showed that the overall performance of the test was improved by using Running buffer II which contained formamide but the specificity of the test remained unchanged, meaning that this strategy was unsuccessful.
However, after testing LFA test created using rpoC gene we saw that the specificity improved. The test was able to positively identify only F. psychrophilum DNA samples. Even though we had a properly working test for F. psychrophilum we ran into a third problem - the test created for F. columnare was also not specific enough.
3rd iteration
3rd iteration
research & imagine
As mentioned, the test created to identify F. columnare 16S rRNA gene was non-specific. It correctly differentiated between F. psychrophilum and F. columnare but was unable to differentiate between Flavobacterium genus and E. coli. To combat this issue we researched more and found out that cslA gene can be used for F. columnare identification as well 11. We imagine that the LFA test created using cslA gene should be of greater specificity.
design
We created ssDNA probes for hypothetical protein B0A56_04620 (Flavobacterium columnare, NBRC 100251 = ATCC 23463) because we found it to be homologous to cslA protein sequence. These probes were also designed to have a greater GC% and length to improve hybridization reaction (BBa_K3416113, BBa_K3416114, BBa_K3416115).
build & test
However, ssDNA probes were created only in silico. Due to the pandemic that affected the whole world our team was unable to test the specificity of the newly designed probes in the laboratory. For this reason we encourage future iGEM teams to test and if needed improve these lateral flow assay probes for F. columnare.
Since LFA requires a lot of optimization our software tool - OnFlow, based on a mathematical model, will be of immense help for future teams in deciding how to build and optimize their LFA tests. OnFlow will determine where to spray capture and control probes and the exact amount of analyte needed to obtain the most visible results on the strip. This software will help to save time and reagents.
learn & improve
What we learned from developing lateral flow assay tests was that a small fragment of 16S rRNA gene is not enough for accurate Flavobacterium species identification. Even though it is routinely used for phylogenetic analysis12. Also, during the LFA development process, we gained new knowledge and skills that will be used in a further scientific career.
1st iteration
Helimerase
1st iteration
research & imagine
After we decided to base our test on nucleic acid hybridization via LFA method we started to search for a suitable isothermal ssDNA amplification method, which could be as sensitive and accurate as possible. When we came upon isothermal helicase dependent amplification (HDA) method, we started to precisely plan our experiments. However, during the planning process, our team realized that the usage of commercial HDA kits rises the price of the test.
During literature analysis we have found a significantly cheaper alternative for the HDA assay. The solution is a bifunctional protein complex called helimerase. Helimerase, according to literature, showed the ability of amplifying significantly longer fragments during HDA reaction13.
Since helimerase complex could be formed in vivo and in vitro, it became crucial for us to find the best conditions of these proteins synthesis and purification. Firstly, based on the research1, we were decided to clone TteUvrD and BstPol into different plasmids. However, after integrated meeting with dr. Giedrė Tamulaitienė, we decided to clone both genes into pETDuet-1 vector. This change of direction should help to minimise possible failure risks as much as possible.
design
The cloning design of the helimerase complex was based on pETDuet-1 vector, which is created for the co-expression of two target genes due to its multiple MCS.
build & test
For the development of such plasmid we performed two steps cloning. During the first cloning, in the MCS-1, WZA2-L1-TteUvrD was fused with 10xHisTag and Maltose Binding Protein (MBP) in its N terminal. During the second cloning WZB1-L1-BstPol was fused with StrepII tag. All these cloning steps were successful, however, during co-expression in E. coli BL21 (DE3) strain, only soluble TteUvrD protein was successfully synthesized.
learn & improve
After result analysis we concluded that the reason why there was no BstPol synthesis could be related with the differences on its copy numbers. Even if pETDuet is low copy number plasmid, 15-20 copies per cell could be too much to translate both recombinant proteins properly.
In order to tackle this issue we have decided to clone the BstPol gene into another plasmid, thus rejecting the idea to perform co-expression from one plasmid.
redesign
As synthesis of TteUvrD was successful, we have decided to use the same construct for further experiments.
However, after we concluded to change the cloning strategy of BstPol, firstly we needed to find the most suitable plasmid for co-expression. For this purpose we had two main criteria – plasmid copy number must be lower than 15-20 and it should have a compatible ori site with pETDuet. One such plasmid is pACYC, which copy number reaches only 10 copies, and its ori site is compatible with pETDuet14.
build & test
After BstPol was cloned successful into pACYC plasmid, we performed its synthesis in E. coli BL21 (DE3) strain. Regarding to TteUvrD and BstPol high-solubility, we were able to purify high amounts of both proteins, so no further optimization for protein synthesis was needed. Nonetheless, protein co-expression and fusion in vivo by using these two compatible plasmids was still unsuccessful.
learn & improve
Despite many attempts, further investigation on the recombinant proteins co-expression is needed. Cloning strategy could be improved by swapping TteuvrD and BstPol places in pETDuet or pACYC MCS. Also, different types of plasmids could be examined in order to find the best options for helimerase formation in vivo.
Treatment
1st iteration
AI-2 Synthesis
1st iteration
research & imagine
By analyzing data, we came up that Flavobacterium form the biofilm, and as well as E. coli use the same autoinducer 2 (AI-2) molecule for quorum sensing. It is a universal „language“ for interspecies communication in bacteria 15-17. This AI-2 molecule or its precursor, 4,5-dihydroxy-2,3-pentanedione (DPD), can be used for manipulating genetic circuits18, but pure AI-2 is costly ( 1mg for 175$). The research goal was to find how we can synthesize it in vitro and use this AI-2 molecule for the following experiments19-20. To reach this goal, we decided to make AI-2 ourselves by using its precursor S-adenosyl-homocysteine (SAH) and two recombinant proteins - LuxS and Pfs (Fig. 1).
Figure 1. AI-2 synthesis from the precursor S-adenosylhomocysteine (SAH)
design
LuxS and Pfs genes were cloned into pET28a(+) vector and synthesized in E. coli BL21 (DE3) strain. Both proteins were purified successfully by using a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden).
build & test
The purified proteins were used for the reaction mixture according to Wattanavanitchakorn et. al. 201419. The synthesized DPD, a precursor of AI-2, was identified with Ellman’s reagent (5,5'-dithiobis-(2-nitrobenzoic acid). This chemical compound is used to quantify the concentration of thiol groups in sample21 (Fig. 2).
Figure 2. The general scheme of Ellman’s reagent reaction with compounds that have a thiol group. The thiol group cleaves the disulfide bond and the cleaved 2-nitro-5-thiobenzoate (TNB−) ionizes to the TNB2−. This ion gives a yellow color to a solution.
learn & improve
However, due to the small amount of the DPD we were not able to use this reagent in further experiments. These results led us to think that SAH quantity was too low for this reaction. To increase DPD concentration, we decided to change proteins’ concentrations and the amount of SAH.
2nd iteration
2nd iteration
research & imagine
After the first trial, we decided to check what else can be done. We have found out that proteins into the mixture should be added one by one. This makes the measurement of the kinetic parameters of each protein much more precise.
design
The Pfs and LuxS protein concentrations’ values and SAH mass were changed.
build & test
The first reaction mixture consisted of everything despite the LuxS. One hour later, LuxS was added. This testing helped to improve the concentration of DPD (Fig. 3), an AI-2 precursor, and gather more information about the enzymatic activity of proteins.
Figure 3. The spectrum after the reaction with Ellman’s reagent. The peak at 412nm identifies dianion TNB2−. The following calculations for determining the AI-2 concentration were done as a protocol requires.
learn & improve
Our experience with Pfs and LuxS proteins showed that every iGEM team that is willing to link their project with a quorum sensing type II system, can easily and cheaply synthesize AI-2 by themselves.
1st iteration
Exolysins
1st iteration
research & imagine
Bacteriophages (phages) are bacterial viruses that can only infect bacteria and are the most numerous biological entities on our planet22. Bacteriophages use exolytic proteins to penetrate the bacteria capsule23. The scientific literature showed that we can use only phage proteins24. We decided to use only phage’s exolytic protein because it still fulfills its function. Thus, the main goal was to find Flavobacterium phage proteins - exolysins. This exolysin would be a part of the genetic circuit that will be induced by AI-2.
design
When the design phase started, we found out that marine phages (e.g. Synechococcus phage) are not as well characterized or do not have representative genomes as mostly known non-marine phages(EBI phage database) 25. This lack of information would lead to problematic experiments.
research & imagine
The project seeks to solve the problems that are caused by Flavobacterium spp. and we had been considering using phages for getting rid of pathogenic bacteria. The bacteriophages have a broad range of specific bacteria lysing proteins, called exolysins. We have determined to find those proteins but the research has shown that there were no homologous Flavobacterium exolysins when the ORFs were compared. Then we were looking for alternatives and consulting with our PI prof. Rolandas Meškys. Then, it was decided to choose the Klebsiella pneumoniae KV-3 phages due to easy accessibility.
design
The exolysin is the main part of the whole gene circuit construction (Fig. 4), which is initiated by AI-2 induced lsrACDBFG(BBa_K3416000) promoter. We had a variety of Klebsiella pneumoniae KV-3 phage depolymerases to choose from. We could have settled to construct all genetic circuits with these depolymerases, but to save time we needed to examine the enzymatic activity of these proteins.
Figure 4. The general genetic circuit of exolysin and endolysin is based on quorum sensing Type II molecule AI-2 inducibility
build & test
After enzymatic analysis, the exolysins called gp529(BBa_K3416029) and gp531(BBa_K3416031) have been chosen for further treatment strategy development. After proteins’ purification, the enzymatic activity was checked with Klebsiella pneumoniae KV-3 biofilms with the spot test. However, the gp529 showed no depolymerase activity, whereas gp531 was successfully purified as an active form.
learn & improve
To successfully destroy pathogenic bacteria biofilm we need an exolysin with good enzymatic activity. The gp531, due to the most efficient enzymatic activity, was chosen to be used for the following experiments.
2nd iteration
2nd iteration
research & imagine
The following task was to make the genes from a genetic circuit compatible that could be used for other iGEM projects. The sequence should be compatible with the most-used assemblies in the iGEM library. After deeper analysis, it was seen that the gp531 native sequence has many repetitive restriction sites. This would have aggravated the construction’s tasks. Thankfully, gp533 exolysin has depolymerase activity and no repetitive restriction sites.
design
The gp533 exolysin was used for a genetic circuit, that is enhanced by AI-2, construction.
build & test
The genetic circuit construction took many attempts to accomplish. Due to the pandemic, it took a lot of time to obtain the exolysins genes and the selection of the most suitable depolymerase took too long to engineer the genetic circuit for our project.
learn & improve
Despite these problems, the exolytic protein showed that it is capable of lysing the pathogenic bacteria and could be used for other teams focusing on alternative strategies of pathogenic disease treatment.
1st iteration
toxin-antitoxin
1st iteration
research & imagine
The treatment section development arose on how we can ensure that genetically modified bacteria could initiate its lysis. The research has shown that there are commonly used so-called kill-switches that ensure that genetically modified organisms won’t be able to transfer their genetic material. To add more, iGEM the main priority is to guarantee that GMOs are safe for usage, so this was an encouragement for us to create another treatment system that includes a classic kill-switch - MazF and MazE26,27.
The iGEM parts library contains MazE and MazF protein-coding sequences but previous teams had uploaded mazF and mazE genes of Bacillus subtilis. After checking these sequences with BLAST, it showed that these sequences code the same genes but of E. coli, not for B. subtilis. Moreover, we decided to compare both treatment genetic circuits and found out the differences between these systems. This might help to establish a better view of how efficient the system is.
Figure 5. The genetic circuit of exolysin and mazEF system
design
Same as before, exolysin is the main part of this construction. Under AI-2 inducible promoter, mazF is expressed. The difference is that genetic circuit accompany Anderson promoter and mazE gene. Before picking the Anderson promoter of the right strength, we needed to compare these promoters by measuring the fluorescence intensity and OD600.
build & test
As mentioned in the Design Page we are measuring AI-2 promoters of different strengths. We used Anderson promoters as a standard for our inducible promoter measurement. For the measurements these circuits were constructed: lsrACDBF-sfGFP, EP14r-sfGFP, EP01r-sfGFP, J23117-sfGFP, J23110-sfGFP, J23104-sfGFP.
To gain extensive insights about what concentration of AI-2 is best for inducing gene expression in vitro synthesized AI-2 was used for inducing the genetic circuit. The goal is to determine the strengths of AI-2 inducible promotersand the concentration threshold of AI-2.
learn & improve
The first trials gave a clearer view of what Anderson promoter we should use. The results indicate that srACDBFG(BBa_K3416000) and its mutants EP01r(BBa_K3416001), EP14r( BBa_K3416014) are quite weak promoters. Furthermore, as in results are described, the AI-2 concentrations were determined. We decided to narrow the window of concentrations’ range. The following experiments took place effortlessly.
2nd iteration
2nd iteration
build & test
After the first iteration, we set a goal for constructing an inducible kill-switch. Everything was working according to plan. After successfully constructing the genetic circuit the measurements were left to be done. We had difficulties with the accessibility of devices, so it made it pretty difficult for obtaining the right conditions and repetitively of experiments. All in all, the results say everything that you need to know about this toxin-antitoxin system and how it works with the quorum sensing Type II system.
learn & improve
We hope that genetic circuits that contain AI-2 inducible promoters or having a better understanding of the toxin-antitoxin system will help other iGEM teams to develop even more sophisticated models for manipulating gene expression.
Prevention
1st iteration
VHSV
1st iteration
research & imagine
To induce an immune response against Viral Hemorrhagic Septicaemia Virus (VHSV) in fish by using a subunit vaccine, we had to select an appropriate protein. Glycoprotein G from VHSV was shown to be recognized by fish immune cells28. However, it was also shown that if the protein is expressed in E. coli, it does not demonstrate any immunogenic properties, because the proper folding of a glycoprotein is not guaranteed due to absence of glycosylation processes29. Due to this reason, the protein should be synthesised in yeast recombinant protein expression systems, where the glycosylation processes occur. After deeper research, we decided to use one of the most common yeast systems, S. cerevisiae.
design
VHSV glycoprotein G coding sequence was fused with a 6xHis purification tag sequence from the N terminus into a pfX7 vector, as it is usually used for protein expression in recombinant protein synthesis systems 30. As usual, firstly we decided to perform cloning in E. coli DH10B strain. Ligate was first cloned into and purified from E. coli DH10B strain. Then transformed and expressed in S. cerevisiae AH22-214 strain.
build & test
After a lot of attempts of cloning there were no colonies of DH10B cells with the transformed pfX7 VHSV 6His N construct in a plate. We started to troubleshoot these results.
learn & improve
After numerous unsuccessful attempts to clone the VHSV gene into pfX7 plasmid we started to search for reasons of these failures. Since we obtained this plasmid from VU BTI EGI department, we decided to have a consultation with Juta Rainytė, who works with this system. After consulting with Juta Rainytė we got acquainted that one of the main traits this plasmid has is its leaky promoter. Due to this, there is a possibility that E. coli lyses itself due to the toxicity of the expressed proteins. The ligate could be transformed into a bacterial strain, that would be tailored for toxic protein expression.
2nd iteration
2nd iteration
research & imagine
After research we found out that E. coli ArcticExpress strain (DE3), is used to improve bacterial protein expression and solubility31. This strain co-expresses chaperonins Cpn10 and Cpn60, that optimizes the protein folding process. In addition, it naturally lacks the Lon protease, which can degrade recombinant proteins. This strain could potentially handle the protein that might be toxic to other strains and help multiply our plasmid for further experiments. Due to this we decided to perform cloning in this strain.
design
To check if the cloning procedure was unsuccessful due to the leakyness of promoter and VHSV toxicity, the next engineering step was to transform VHSV gene containing plasmid into E. coli pfX7 plasmid and transforming it into E. coli ArcticExpress (DE3) strain. Because this strain possesses an additional plasmid for chaperonin co-expression, it needed to be separated from our plasmid of interest.
build & test
After we made all the required experiments we finally saw that the VHSV cloning step was successful.
learn & improve
Successful cloning procedures in ArcticExpress (DE3) strain led us to the conclusion that the expressed protein due to the leaky promoter was indeed toxic to DH10B strain. Now the resulting plasmid could be cloned into S. cerevisiae.
3rd iteration
3rd iteration
research & imagine
design
The protein can be synthesised in different S. cerevisiae strains and purified via Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden), which is used for purification of recombinant proteins with 6xHis tag. The pH and contents of buffers for purification can be adjusted according to purification results.
build & test
The results of SDS-PAGE electrophoresis indicate that the protein of interest was not synthesised, yet an unknown protein half the size was purified.
learn & improve
The protein synthesis and purification conditions can be improved by:
- Constructing a plasmid with a 6xHis tag on the C terminus of the coding sequence. It might help if 6xHis tag on N terminus is hindered and inaccessible when it is being purified.
- Fusing the glycoprotein G coding sequence with Maltose-Binding protein (MBP) and Tobacco Etch Virus (TEV) Protease coding sequences. MBP tag should help with solubility of the protein and TEV protease site is used to remove MBP in that case if this tag is hindering or inhibiting immune response
- Choosing other S. cerevisiae strains
- Trying Pichia pastoris for protein expression. This idea was proposed by Dr. Rasa Burneikiene-Petraityte, gene engineering expert, by stating that Pichia pastoris is glycosylating less and with more accuracy than S. cerevisiae. However, this strategy would require an extensive amount of time to implement, which we unfortunately did not have in our disposal. It could be used in the further developments of the project.
- Changing the pH of the buffers used for the lysis of yeast and protein purification. By trying and comparing different buffers such as HEPES, Tris-HCl, PBS and changing pH we can determine the most optimal conditions for the protein environment where it would be most soluble and stable.
- Balancing the ionic strength of the protein purification buffers. This might help to achieve higher purity of the specific protein by interacting with the proteins. Only the ones with high affinity to the column can bind to the resin, whereas, protein with less affinity to the column will be washed away. Also, ionic strength might help with the solubility when ultrasound lysis occurs by interacting with protein’s amino acids and hiding hydrophobic regions.
1st iteration
Gldj
1st iteration
research & imagine
Since our project is mainly focused on Flavobacteria caused diseases, we paid a lot of attention for the creation of an oral subunit vaccine against columnariosis disease, caused by Flavobacterium columnare. We found that this bacteria contains Gliding motility protein (GldJ), which is located on the surface of F. columnare33. Previous research showed that this protein could induce an immune response in fish since GldJ has well-exposed epitopes and is recognized by fish immune cells34.
design
We decided to clone the GldJ coding sequence into pET28a(+) vector, as it is commonly used for recombinant protein high-level expression. Due to huge availability to E. coli strains we have chosen to optimize induction conditions. We decided to try three different strains for their intrinsic properties of protein expression:
- BL21 (DE3) strain allows high-efficiency protein expression.
- Rosetta (DE3) contains rare tRNAs that might be needed for our target protein.
- ArcticExpress (DE3) has two heat shock proteins which should help with protein folding.
build & test
After SDS-page gel analysis we saw that protein was induced but there were 2 protein bands that were not the size we expected. One of the induced protein sizes was bigger by 5-10 kDa, while the other was smaller by 5-10 kDa. We concluded that most of the protein was in the insoluble fraction, that is why there was no protein detected after purification with a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden). Induction worked best in ArcticExpress strain but the protein was mostly in the precipitant.
learn & improve
When we got unexpected protein bands, it was decided to check whether the gene has any mutations. Sanger sequencing revealed that there were none. We thought that it would be great to purify at least a little bit of the desired protein if protein solubility would increase.
2nd iteration
2nd iteration
research & imagine
After carrying out literature analysis we found that trehalose can enhance the protein solubility by stabilizing it. Trehalose serves as an osmolyte and prevents protein aggregation. It was hypothesized that it might contribute as a chemical chaperone, that is shown after inspecting change protein folding rates35.
design
It was decided to add trehalose into a resuspension buffer which is used for biomass dispersion before ultrasound lysis.
build & test
SDS-page showed that there are more proteins in soluble fraction when trehalose was added. Unfortunately, when we performed Western blot analysis after protein purification, there were no detectable proteins in our elution samples.
learn & improve
Next steps could be:
- Utilizing sonification (resuspension) buffer.
- Trying other E. coli strains. For example: Hms174(DE3) - an alternative to BL21(DE3); Origami, which employs chaperones; Nova Blue (DE3) intended for toxic genes (we speculate, that maybe the correctly folded protein is toxic for bacteria).
- Cloning the gene into other vectors. Changing the vector with a weaker promoter (not T7), could improve protein folding, since in pET28a(+) it could happen too fast.
- Changing and evaluating results with various inductor (IPTG) concentrations.
- Different tag such as MBP should solve the solubility problem.
- Purifying protein under denaturing conditions (with urea or guanidine hydrochloride) might also help.
1st iteration
alginate beads
1st iteration
research & imagine
If the immunogenic protein is administered orally, it has to reach the gut of the fish to induce an effective immune response36. In order to achieve this, the proteins have to pass the stomach without any degradation. Scientists have demonstrated that calcium alginate was not digested by stomach enzymes and could only be degraded with alginate lyase produced by specific bacteria, for example Klebsiella pneumoniae or Pseudomonas maltophilia37. Such kind of bacteria could be found in the midgut of the fish, thus making a perfect place for alginate degradation. This feature of alginate degradation could be exploited by enveloping the immunogenic proteins in it.
design
The immunogenic protein GLDJ was mixed with alginate and dropped into a calcium chloride bath to form a bead38.
build & test
Expressed proteins were purified by using a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden), which was used for recombinant proteins with 6xHis tag purification, and enveloped in calcium alginate. In order to see whether proteins stay stable after the encapsulation process, the beads were examined in vitro. Therefore, a droplet of digestive enzyme (trypsin/pepsin) or a buffer of an appropriate pH was applied directly onto the bead and incubated for up to 2 hours. The resulting changes in the properties of color and solidity of the bead were observed.
learn & improve
Alginate can effectively protect from the stomach acidic environment and should protect from alkaline pH. It is important to know if the immunogenic proteins (GLDJ) are spread throughout all the beads in a uniform manner. This ensures equal distribution of the immunogens in every bead. However, it was hard to determine that from the assay. Moreover, the changes after treatment with enzymes and buffers are too minimal to draw substantial conclusions about the integrity of the beads. In order to improve the tests of protein distribution and digestion, we decided to first optimise it with a visually observable protein. After these steps, beads production method can be applied to the actual immunogenic proteins.
2nd iteration
2nd iteration
research & imagine
Green fluorescent protein (GFP) is a commonly used protein for visualization of processes that are hardly visible. GFP could be encapsulated in calcium alginate and observed to determine the uniformity of the protein throughout the bead and its changes after the application of enzymes or buffers. The conditions of the encapsulation process could then be adjusted with the control beads and applied when making vaccines with immunogenic proteins.
design
GFP coding sequence cloned into a pET28a(+) vector was expressed in E. coli BL21(DE3) strain. After protein expression and purification, the protein was mixed with alginate and dropped into a calcium chloride bath to form a bead.
build & test
Expressed GFP protein was purified by using a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden) and enveloped in calcium alginate. The uniformity of protein distribution in different beads can be easily assessed. GFP beads can be used to easily determine if an alginate bead would be degraded by digestive enzymes over time. The changes can be seen in mere seconds with the naked eye.
learn & improve
The results of the tests had shown that the beads did not degrade in the presence of digestive enzymes. Yet, it was crucial to determine if they would release the protein in the midgut of the fish.
3rd iteration
3rd iteration
research & imagine
In order to determine this, alginate lyase should be present in the environment of the bead. Bacteria species that have this enzyme are Bacillus circulans, Klebsiella aerogenes, K. pneumoniae, Pseudomonas maltophilia, P. putida and P. aeruginosa. We wanted to use Pseudomonas sp., since it is able to synthesize alginate lyase and it lives in the midgut of rainbow trout37. However, due to the pandemic and this bacteria pathogenicity for humans, we could not acquire it for the testing. Fortunately we found that Klebsiella pneumonia KV-3, which was used in other parts of our project, is also able to synthesise the lyase 39. Hence, it could be used to validate if the alginate beads do, in fact, degrade in the presence of this enzyme.
design
An exact number of uniform sized alginate beads with GFP were dropped into a flask with Klebsiella pneumonia KV-3 and grown for 3 days. We dropped the same amount of beads into a flask with E. coli BL21 (DE3) strain as a control. Also, we used alginate beads that lack the protein which was also used as described above with GFP alginate beads.
build & test
After 3 days, the experiment showed that our Klebsiella pneumonia KV-3 strain really does have alginate lyase because all beads (with and without GFP) were decomposed while the ones in E. coli flask were still intact. The only question was why did it take exactly 3 days?
learn & improve
This might be due to biofilm forming after several days when the rpm is low. Also, the medium might have a big role here. This might be due to catabolite repression mechanism where bacteria prioritize metabolising most nutritious substances (the ones which give the most energy) inside the LB medium and only after they are malnourished then bacteria begin secreting alginate lyase or other proteins into the environment. In order to prove our hypothesis, further experiments would be:
- Trying different temperatures. Not applying optimal conditions might have an influence on protein expression.
- Testing if the bead maintains its integrity while being spun at a higher rate.
- Trying different medium from something minimal like M9 to enriched, such as blood medium or chocolate-blood medium:
- M9 would be beneficial, because it has very low autofluorescence and we could use plate reader assay to determine when the GPF in the beads dissolve.
- On the other hand, Blood medium is suitable for pathogenic bacteria (our Klebsiella pneumonia KV-3 is pathogenic as it was cultivated from veterinary isolate). This is why the protein expression could be different than in LB medium.
4th iteration
4th iteration
research & imagine
The tests confirmed that calcium alginate gets degraded in the presence of alginate lyase. It was proved earlier that the beads do not degrade when trypsin and pepsin separately are present in their optimum environment. The last challenge that the beads need to withstand while traveling through the fish digestive tract is the physical pressure from the peristaltic movements40. According to research 41, the pressure that the bead should withstand to reach the midgut should be between 0,13-0,32MPa. In accordance to this, the beads should be tested to see if they are strong enough.
design
To ensure the reliability of the testing results, beads of a uniform size were selected. They were compressed using a universal testing machine (TIRA test 2300) until rupture.
build & test
The results of the tests indicated that the beads could only withstand a pressure a few times lower than recommended. Also it was clear that the presence of the protein in the bead increased the integrity of it around 20%.
learn & improve
These steps could be taken in order to make the alginate beads stronger and more implementable:
- The beads could be kept in CaCl2 solution after production for a few days, because alginate tends to gradually harden overtime when exposed to CaCl242.
- The size of the bead could be varied between 3mm to 5mm to see which one is the strongest. This should be done keeping in mind the change of protein concentration in a single bead, to ensure the correct dosage of vaccines for immunization.
- The alginate solution concentration could be increased.
- The concentration of the protein in an alginate-protein mixture could be increased, as it tends to increase the integrity of the bead as observed earlier.
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