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LabBook

Facing back to the Etendard glacier, in between the peaks of Maurienne and the ones of Oisan.
Credits: Estelle Vincent

Step 1 : Target Preparation

The first rate-limiting step in the detection of Vibrio Cholerae is the extraction of its DNA. To this aim, the bacteria has been deeply studied, as well as the current DNA extraction techniques.

1. Vibrio Cholerae

1.1 Classification & Generalities

Table 1 : V. Cholerae classification
Domain	Bacteria
Phylum	Proteobacteria
Class	Gammaproteobacteria
Order	Vibrionales
Family	Vibrionacea
Genus	Vibrio
Species	Vibrio cholerae

V.Cholerae is a thin gram-negative proteobacterium that has a flagellum which gives it mobility [2]. This bacteria is responsible of cholera disease, causing severe contagious epidemia. This bacteria use to grow in basic conditions (Optimal growth pH : 9) [1] with 1-3% NaCl in liquid or solid mediums [3].

1.2 Growth in laboratory

Aeroanerobic bacteria grown on conventional media. Optimum growth in medium with 1 to 3% NaCl pH 9, in liquid media (Colonies in 3-4 h at the surface) or in solid media (colonies in 8-10 hours). The bacterium can also grow on bile salt media. According to these characteristics, alkaline peptone water pH 8.6 3% NaCl can be used as an enrichment medium as well as an alkaline agar pH 9. Colonies are 2 to 3 mm in diameter and are smooth, Flat and transparent. [3]

1.3 Tanks and contamination

Main V.Cholerae tanks are humans and dirty water, it seems that global warming is creating favorable conditions to this bacillus [4]. Even if there are many kinds of this bacteria, only 2 serogroups are directly responsible of Cholera : O1 and O139.

Human transmission is linked to inappropriate access to clear water. This bacteria can survive more than 15 days in water. Contaminations are also possible with contaminated food like vegetables or fishes, The infectious dose is between 106 and 1011 vibrios ingested [5]. The infectious dose depends on gastric acidity (the lower the acidity, the fewer vibrios required to cause infection)[6]

1.4 Physiopathology & Virulence

Cholera is a very virulent disease that can cause severe acute watery diarrhea. The bacillus can be found in patient’s stools for 1 to 10 days after infection (106 to 108 bacillus/mL [3], [5]) and is disposed of in the environment where it can potentially infect other people. An untreated choleric person would produce 10 - 20 liters of diarrhea a day [7]. The cholera toxin (CTX) is an oligomeric complex made up of six protein subunits responsible to the symptoms. Once inside the cell, the A1 subunit is freed to bind with a human partner protein : Arf6 [8]. This bound exposes its active site, allowing it to ribosylate the Gs alpha subunit of G protein. This results in constant cAMP production, which leads to the secretion of water, sodium, potassium, and bicarbonate into the lumen of the small intestine and rapid dehydration.
A healthy human feces contains 1012 bacterias per grams, more than 400 different species can be found [3]. V.Cholera is more in patient’s feces than other bacterias [3] [9].

The gene encoding the cholera toxin was introduced into V.Cholerae by horizontal gene transfer. Virulent strains of V.Cholerae carry a variant of a bacteriophage called CTXφ[10].

Figure 1 : Infection of Vibrio cholera by the CTX bacteriophage

2. DNA Extraction

In order to obtain the target, the vibrio DNA must be extracted.

2.1 Laboratory extraction

Nowadays, DNA preparations are widely used because of their easy using. A lot of suppliers proposes kits (NEB, QIAGEN…). Kits allow to extract 50 ug to 10 mg of DNA thanks to different protocols/materials (Miniprep, Midiprep, Maxiprep, Megaprep and Gigaprep [11]) and different kind of polynucleotides can be extracted : Plasmidic DNA, RNA or even genomic DNA for instance. Protocols can also be more or less fast, depending on the the supplier’s technology.

2.2 Paper-based technology

A famous field of research consists in paper-based extraction technology. It permits low-costs diagnosis. The point is that these technologies permit easier way to perform sample preparation. For example, it is possible to make an automated DNA extraction from the human whole blood in only 7 minutes [12].
Some scientists also developed an automated way to proceed DNA extraction : it combined magnetic beads, paper, stepper actuators and a micro-computer called Arduino. [13]
Thanks to that technology and using 96 well plates, it is possible to target the apicoplast genome for malaria diagnosis [14].

2.3 Isothermal amplification - LAMP

Loop mediated isothermal amplification (LAMP) is an isothermal technique for the amplification of DNA [15] widely used in point-of care diagnosis because it is cheap and simple[16] [17]. The sensitivity can reach 92% [17] making it a serious technology for malaria diagnosis.
Combined with paper-based technologies, it is possible to develop low-coast paperfluidic molecular diagnostic chip that can extract, amplify and detect DNA from clinical samples in less than 1 hour even in resource-limited settings [18].

3. Vibrio Cholerae extraction

3.1 Vibrio Cholerae sensitivity

To lyse bacteria, several disinfection solutions were prepared, such as 2-5% phenol ; 1% sodium hypochlorite ; 4% formaldehyde ; 2% glutaraldehyde ; 70% ethanol ; 70% propanol ; peracetic acid To 2%, to the hydrogen peroxide to 3-6% and to the iodine to 0.16% [19]. As mentioned before, V.cholerae is not supposed to survive in acid mediums [6]. Another possibility to kill it is to create a thermal shock (0°C)[20]

3.2 Lysis buffer

After many intern discussions, DNA amplifications were dropped because the bacteria is overexpressed in the sample. Lysis buffer is used to extract DNA from bacterias. It contains Urea 4M and DNAse inhibitors. Urea 4M allows a lysis efficiency of 90% in less than 10 minutes [21], which is fast and interesting given the fact that V.cholerae is overrepresented in patient’s faeces [3] [5].

3.3 Protocols and Results

Late exponential phase cells of V.Cholera will be harvested and resuspended in 20 mL of 4 M-urea with DNAse inhibitors. The following step is an incubation of 10 min at 24°C, by which time more than 90% lysis occurs. The only difference is that we extracted the DNA from E.Coli instead of V.Cholerae for security measures.

The following protocols (Table 2) has been done in laboratory in order to check if the centrifugation can be removed during the DNA extraction. Two different elution buffers are compared.

Table 2 : Resume of the protocols used in laboratory to test this process of DNA extraction
Test A Silica column + TE elution buffer	Test B Silica column + elution with distilled water
Insert 20uL of PK in a 1,5mL eppendorf	Insert 20uL of PK in a 1,5mL eppendorf
Add 200 uL of sample + 200 uL of PBS 1X	Add 200 uL of sample + 200 uL of PBS 1X
Add 200 uL of AL buffer + shake for 15 sec	Add 200 uL of AL buffer + shake for 15 sec
Incubate for 20 min at RT	Incubate for 20 min at RT
Add 200 uL of 96% ethanol + shake for 15 sec	Add 200 uL of 96% ethanol + shake for 15 sec
Drop the mix in the column Elute (using the piston) → Discard the eluant	Drop the mix in the column Elute (using the piston) → Discard the eluant
Add 500 uL of AW1 buffer Elute (using the piston) → Discard the eluant	Add 500 uL of AW1 buffer Elute (using the piston) → Discard the eluant
Add 500 uL of AW2 buffer Elute (using the piston) → Discard the eluant	Add 500 uL of AW2 buffer Elute (using the piston) → Discard the eluant
Add 200 uL of TE buffer	Add 200 uL of distilled water
Incubate 1 min Elute (using the piston)	Incubate 1 min Elute (using the piston)

The use of the piston is the most important step. Indeed, it enables to replace the centrifugation and also to reduce the extraction time. The piston needs to have exactly the same diameter than the column. It is inserted in the column and pushed to exert a pressure on the mix, allowing the liquid to pass through the silica.

Figure 2 : Explanatory scheme of the use of the piston during the experience

NanoDrop measures have been done, giving the following results :

Table 3 : Table of the DNA concentrations in ng/uL obtaining at the end of the protocol
Test A	Test B
28.8	19

Finally, extracting genomic DNA without centrifugation is possible. The concentration is higher when the elution is done with the TE buffer. This concentration range allows the pursuit of the experience.

3.4 Preparation of the target

The next step is the cutting of the Vibrio DNA by Alu I: one hour at 37°C in CutSmart Buffer. Then a denaturation is done at 73°C. The target now ready to be detected and the next step, the detector activation, can occur.

Step 2 : Construction of the plasmid detector

1. Design of the detector in silico

1.1 The target

The bioinformatic work aimed to find a specific Vibrio cholerae sequence to detect. First, it was necessary to study Vibrio cholerae (Vc) pathogenicity. The known epidemic strains [1] O1 (GeneBank : KF664566.1) and O139 (AF302794.1) sequences available on PubMed have been deeply studied. Importantly, Vc’s pathogenicity is premised on the integration of a bacteriophage sequence - bacteriophage CTX - in Vc’s genome [2]. Indeed, the integrated sequence contains genes for toxins, responsible for symptoms of cholera disease. Based on the actual knowledge [3], the target has been chosen inside CTX’s genome, and more precisely within the non-pathogenic genes of CTX (i.e. genes with roles in the reproduction or in the metabolism of the bacteriophage). One requirement was to detect something specific to the bacteriophage CTX. After a blast of all CTX’s genes, genes with low similarity to other organisms have been retained. Then the target had to be quite small (less than 100 bp) as well as being cut with a single enzyme (ideally blunt end). Finally, the target was found in RstA gene (1080bp, implicated in CTX DNA integration and replication [3]), between nucleotide 726 and nucleotide 765.

1.2 The probe

Two versions of the probe has been designed. The first (Version 1) permits a cloning with a single restriction enzyme EcoRI (Figure 2).

The insertion strategy was changed afterall in order to facilitate the cloning.

The probe has been imagined based on the genius system of Cork Ireland 2015 team and all the data furnished by New England Biolabs (NEB) website. It is flanked with two EcoRI sites at each end for its insertion in a plasmid backbone. It also contains :
Two restriction enzymes producing cohesives end, BmtI and BglII, which goal is to remove the little sequence in between on the bottom strand and thus to create a perfect complementarity with the target.

Two nicking enzymes, Nt.BspQ1 and Nb.bts1, i.e. enzymes that cut one strand of the double DNA strand. Thereby, the top strand is removed, allowing the binding of the target.

1.2 The plasmid backbone

The plasmid carrying the detector probe has to meet at least the following conditions:
have a red fluorescent reporter: RFP or mCherry (necessary for the final detection)

have no restriction sites used for probe activation
To find a correct plasmid, the iGEM 2017 DNA distribution kit has been fully screened and different parts have been tested in the lab. The perfect backbone appeared to be the biobrick J04450. The process to find this biobrick is explained right below.

2. Construction of the detector in practice

2.1 Tests for the choice of the plasmid backbone

2.1.1 Screening of DNA Distribution kit 2017

So, among all the biobricks proposed in iGEM 2017 kit, plasmids were screened in the following precise criteria.
Red fluorescent reporter
Plasmid had to carry a RFP or mCherry gene, because red fluorescence is necessary for the final detection. Finally, all the plasmids retained were carrying RFP gene.
“All-in-one”
That means the reporter system (promoter, ribosome-binding site (RBS), gene, terminator), which codes for RFP protein, is delimited by restriction sites XbaI and SpeI. Thus, the probe can be inserted in the prefix or suffix. In this case, the probe is inserted as a prefix: the first version was inserted in EcoRI site, while the second version was inserted between EcoRI and XbaI.

No restriction sites should be used for probe activation, at least for the double strand enzymes BmtI and BglII. The risk would be to cut the plasmid backbone, then making transformation and detection impossible. Thus, each plasmid map has been digested in silico with BglII and BmtI via Labgenius Mapper, to check the digestion profile.

High level copy, allowing an easier plasmid production.

Properly sequenced, in order to be sure of the genetic content used.
Consequently, a few interesting biobricks were selected.

2.1.2 Biobrick candidates & tests

A serie of biobricks with constitutive and inducible promoters was selected. Even though LacI induction appeared to be a better choice in regards to its well-documentation, biobricks were tested with either constitutive promoter or LacI inducible promoter.

Table 1: 4 post-screening candidates to be the plasmid backbone for the detector.
BBa_K608014	BBa_K608017	BBa_J04450	BBa_K801100
Constitutive promoter	Constitutive promoter	Inducible promoter (IPTG)	Inducible promoter (IPTG)

For the constitutive promoters, biobricks designed by iGEM Freiburg 2011 were chosen. Based on their research, BBa_K608017 and BBa_K608014 seemed to be the most appropriate parts in our case because of their high level of fluorescence. Concerning the inducible ones, BBa_J04450 was selected for its consequent documentation and BBa_K801100 chosen randomly among the list of biobricks producing RFP.

Choice of the promoter
In this experiment, the four plasmids were transformed in parallel in competent DH5ɑ, which were then incubated overnight in a 5 mL pre-culture (LB+ chloramphenicol). Inducible promoters were incubated with 7 different IPTG concentrations overnight (1 CFU per condition). The day after, 10µL of overnight pre-culture were dropped on a microscope slide in order to observe fluorescence thanks to an epifluorescence microscope. No fluorescence could be observed in the tubes with bacteria transformed with biobricks having constitutive promoters. There is a high probability that these plasmids need more time to emit observable red fluorescence. In contrast, the two biobricks with inducible promoters showed fluorescence. After centrifugation, bacterial pellets were definitely fluorescent under natural and UV light (fig 7).

Step 3 :

Our Results