Team:TU Kaiserslautern/HowToLeishmania

Introducing Leishmania How to Culture Dilution Preparing Cultures Induction Transfection Measurements Transfection Success Protein Production Further Experiments

Leishmania are relatively unexplored organisms in biotechnology. However, its abilities to alter proteins post-translationally, similar to humans, are remarkable [1]. For a long time this organism have been given too little attention, which we aim to change. Normally Leishmania are human pathogenic organisms, which usually makes it difficult to work with them. The strain Leishmania tarentolae, however, is apathogenic to humans. As an S1 organism, it can be safely worked with in the laboratory, which makes it more than equal to current model organisms such as E. coli, Chlamydomonas reinhardtii and yeast cells. Moreover, much like bacteria, algae and yeast cells, they can be kept in large numbers in bioreactors.

What fascinates us so much about the Leishmania is that the ability of human-like post-translational modification is not present in the current model organisms and thus one can directly use the proteins of these organisms for medicine or pharmaceuticals. In addition, Leishmania are easy to handle, which makes working with them very pleasant.

Glycosylation patterns of diffrent organisms[2]

Introducing Leishmania tarentolae

Leishmania tarentolae are eukaryotic parasites that use small reptiles as hosts. They form a genus of organisms belonging to the protozoan group. As obligate parasites, they depend on a host organism to reproduce and grow.

Leishmania tarentolae[3]

In order to keep them in liquid culture, there are a few things to keep in mind when dealing with Leishmania.

To make this a little easier for subsequent teams, we are writing a how to handle.

How to Culture Leishmania

The Raising of the Cultures

We kept our cultures at a temperature of 27°C in the incubator. The optimum is 26°C. If the temperature rises, the growth of the cultures slows down and at a temperature of 37°C the parasites die. Leishmania tarentolae must be cultivated under aerobic conditions. Therefore, cultures are incubated on a shaker at approximately 53 rpm.

For uniform growth it is important to always add new medium to the culture, or to remove dead cells and old medium. The generation time of Leishmania tarentolae is about 7-9 h. In the stationary phase the culture is at a cell density of 0.9-2* 10⁸ cells/ml.

For the cultivation different media or substances are necessary. Quantities always refer to 1 L medium. In addition, after autoclaving the media at 120°C for 15 minutes, the following antibiotics are added depending on the media: Nourseothricin (NTC) 100 mg/ml[4] , Bleomycin (Bleo) 100 mg/l[5], Hygromycin (Hygro) 100 mg/ml[6] , Tetracycline (Tet) 10 mg/ml[7] . For a final concentration of 5 μg/ml of hemin[8] , add 2 ml of 500x stock solution of hemin to 1 L of medium.

BHI medium:[9]

37 g BHI (brain heart infusion)
hemin (1:500)
1 L millipore water

Medium for initial cultures:[9]

37 g BHI (brain heart infusion)
Hemin (1:500)
Antibiotics (NTC (1:1000), Hygromycin (1:1000), PenStrep (1:200))
1 L millipore water

Selection medium:[9]

37 g BHI (brain heart infusion)
Hemin (1:500)
Antibiotics (NTC (1:1000), Hygromycin (1:1000), PenStrep (1:200), Bleomycin (1:1000))
1 L millipore water

Medium for the induction of gene expression:[9]

37 g BHI (brain heart infusion)
Hemin (1:500)
Antibiotics (NTC (1:1000), Hygromycin (1:1000), PenStrep (1:200), Tetracycline (1:1000), Bleomycin (1:1000))
1 L millipore water

Medium for freezing cultures:[9]

37 g BHI (brain heart infusion)
300 ml glycerol
700 ml millipore water

Medium for BHI agar:[9]

37 g BHI (brain heart infusion)
0.8 g folic acid
8 g agar

Transfection buffer:

21 mM HEPES
137 mM NaCl
5 mM KCl
0.7 mM NaH₂PO₄
6 mM Glucose

The Dilution of the Cultures

It is important to dilute the cells sufficiently. If the cell density exceeds 10⁸ cells/ml, the culture may die.

For us, dilution on Mondays (1:10), Wednesdays (1:10) and Fridays (1:20) proved to be efficient. In this way, we were able to maintain the cell density of our cultures at about 0.5-0.7* 10⁷ cells/ml throughout.

Preparing Cultures for Experiments

The first step is to determine the cell density of the culture. For this purpose, a fixing medium mixed 1:2 with liquid culture is used.

A Neubauer counting chamber is used to count the cells. In the microscope, 40x magnification should be set.

The small squares (4x4) in the center of the counting chamber form a compartment with the dimensions 0.1mm x 0.2mm x 0.2mm. This gives a volume of 4nL.

10 of the 4nL subjects should be counted. The mean value is then formed from the counted values.

As an example, if an average of 32 cells per compartment was counted, the cell density would be 32 cells/4nL. To arrive at cell density/nL, divide by 4. To get the cell density/mL you need to multiply with 10⁶.

For our example this results in:

\[\frac{32\;cells}{4} \cdot 10^6 = 8\cdot10^6 \frac{cells}{ml}\]

Afterwards, the dilution of the fixing solution must be taken into account or included in order to arrive at the final cell density/ml.

Induction with Tetracycline

Induction of protein expression is controlled by the T7 expression system. Leishmania constitutively express the tetracycline repressor and the T7 RNA polymerase. The repressor binds to the operator, inhibiting transcription of the T7 RNA polymerase.[9]

Induction system of the T7 promoter system in Leishmania tarentolae. [9]

Gene synthesis is induced by the addition of tetracycline. The genes for the tetracycline repressor and the T7 RNA polymerase are localized on the chromosomal DNA of Leishmania. Both the repressor and the RNA polymerase are thus continuously expressed. In the presence of tetracycline, the Tet repressor binds more tightly to the tetracycline and thus detaches from the operator. Now the T7 RNA polymerase can bind to the promoter and read the desired gene.[9]

For maximum protein yield, cultures are induced with tetracycline (1:1000; at a concentration of 10 mg/ml) two days before use. From now on, these cultures must be diluted with the selection medium containing tetracycline.

Transfection

After the desired construct has been cloned into E. coli using Modular Cloning, we want our Leishmania to produce the desired protein. For this, the construct must be introduced into thel DNA of Leishmania. This is done with the help of electroporation.

Important: The transfection must be performed under the sterile bench.

Needed Materials

Your leishmania original cell culture
DNA – constructs
Distilled H₂O (for the negative control)
2 ml eppendorf tube
Transfection buffer

The information on the transfection buffer can be found below “the Raising of Cultures”.

Day 1

Preparation

To be able to perform a transfection, the required cell culture volume is calculated with a cell density of 10⁸ cells/ml. It should be noted that a rather densely overgrown culture is used for this purpose. It is helpful to check out how the leishmania cultures are doing under the microscope after each dilution. For this purpose, culture is taken and mixed 1:1 with fixing solution in an eppendorf tube.

One calculates the mean value of the counted cells per box (at least three boxes should be counted).
Example:
- Construct count: 7 constructs + 1 negative control
- Counted cells: 88, 94, 100, 84
The mean is multiplied by 0.75 to get to a total of 10⁶ cells. However, since we are working with a cell density of 10⁸ cells, the decimal point is moved forward two more places. \[ 91.5 \cdot 0.75=0.68625\,ml\]
To calculate how many ml of cell culture are needed for the constructs to be transfected, the number of constructs (do not forget the negative control) is divided by the calculated cell density. \[\frac{8}{0.68625}=11.65\,ml\] →Thus 11.65 ml of cell culture is needed for the example transfection.

For further preparation, the DNA constructs must be sterilized in the PCR cycler if they have not previously been processed under a sterile bench. For this purpose, the constructs are placed in the PCR cycler at 95°C for 5 minutes.

Transfection

Remove the calculated cell culture volume and transfer it to an eppendorf tube.
The cells are centrifuged at room temperature (RT) by 3310 g for 5 min.
Take the supernatant off and wash the pellet with 1 ml of transfection buffer.
The cells are centrifuged again at RT by 3310 g for 5 min so you can take the supernatant off.
Add 150 µl transfection buffer per transfection to the pellet.
Put 150 µl cell culture in electroporation cuvettes and add 50 µl DNA (with 5 – 10 µg of DNA) construct.
For a negative control, add 50 µl of distilled water to the 150 µl cell culture in the electroporation cuvettes.
The cuvette with the cell / DNA suspension is placed in the Nucleofector and the transfection is carried out with the program U-033.
The suspension is pipetted from the cuvette into 1 ml BHI medium into an eppendorf tube and incubated at 27 ° C overnight.
→It is important to note that for successful transfection, the BHI medium should be used without any antibiotics (only hemin is added), otherwise the cells will die before the constructs are successfully integrated, as they do not yet possess the selection resistances without the construct.

Day 2

Plating out cultures on a plate

The next day, the transfected cultures must be plated out to allow individual colonies to grow.

Important: Plate preparation and plating must be performed under sterile conditions under the sterile bench.

Needed materials

Transfected cultures in the eppendorf tube
Petri dishes
BHI – agar medium

The information regarding the BHI agar medium can be found below “the Raising of Cultures”.

Preparing the plates

The BHI - agar medium is completely dissolved in the microwave. Care should be taken when opening the lid of the bottle slightly to prevent the bottle from bursting. The medium must be allowed to cool under the sterile bench, to hand warmth.
Then the antibiotics are added:

NTC (1:1000)
Hycromycin (1:1000)
Bleomycin (1:1000)
Hemin (1:500)
FBS (1:10)

If the temperature of the medium is too high, the antibiotics as well as the hemin and FBS may be destroyed.
When everything is mixed, the plates are poured to about 2-3 mm layer thickness.
The plates are left open until the agar has hardened and no liquid is visible.
If the plates are to be stored for a long time, they can be sealed with parafilm and stored in the incubator by 27°C.

Plating out the cells

Centrifugation of the cells for 5 minutes at 3310 g.
1 ml supernatant is taken from the eppendorf tube.
The rest is used to resuspend the pellets and to spread on the plates.
Let the plates dry. When the cells have been plated out, care should be taken that the plates aren’t too dry, but also not too most.
The plates are sealed with parafilm.
The plates are stored at 27°C in the incubator.

After 1-2 weeks, the first colonies should appear.

Next step

Pick a culture from a plate

When the colonies have reached a size of 1-2 mm in diameter, they can be transferred into liquid medium using an inoculation loop. The agar around the culture is cut out with an inoculation loop and transferred into an eppendorf tube with 1.5 ml selection medium. The picking of the cultures must also be carried out under sterile conditions. The culture is incubated overnight in this medium and transferred to a culture bottle in 5 ml of selection medium the next day.

Measurements in Leishmania tarentolae

An important part of the work is to prove that your system works in L. tarentolae. This is crucial for further work. For this purpose, we now present some experiments that will help you to prove the built-in system. First, you should start to find out whether the transfection of the DNA constructs in Leishmania was successful. Here you have the possibility to detect the blecherry marker, present in the inserted vector with the help of a colony PCR and an agarose gel electrophoresis. Furthermore, you can detect a successful transfection with the help of fluorescence detection of mCherry, because the mCherry gen is also located on the transfected vector. At the same time, fluorescence detection demonstrates that the built-in system is functional when mCherry is detected and thus produced by L. tarentolae. Once the successful transfection and the function of the system has been verified, you can go one step further and use immunoblots to detect the production of your protein of interest. In addition to these experiments, the influence of the transfected constructs and the antibiotics used on the leishmania cultures can be analyzed. A simple growth curve can be performed for this purpose.

Detection of a Successful Transfection

Agarose gel electrophoresis

Preparations of the samples for PCR

Important: Sample preparation must be performed under the sterile bench.

2 ml of each cell cultures used are taken and transferred to an eppendorf tube.
Centrifuge the samples at 3310 g for 5 - 10 minutes.
Remove the supernatant completely.
If the samples are not used directly for PCR sample preparation, they can be stored at -20°C.

Lysis of the samples

Thaw the samples if necessary and place them in a heating block at 97°C for 10 minutes.
In addition, some 10% SDS can be added to the samples to lyse the cells.

Now your samples are ready to be prepared and PCR and the following agarose gel electrophoresis can be performed. Depending on the DNA polymerase used, the appropriate protocol must be used.

Spectrum measurement

Preparation of the samples

First, the cells of the individual cultures must be counted in a counting chamber under the microscope and the required cell culture quantity calculated with a cell density of 10⁶ cells/ ml. How to calculate the cell culture amount can be found under “Preparing Cultures for Experiments”. It should be noted that it is useful to prepare triplicates for each sample and to calculate the mean value for analysis. The preparation of cultures for experiments can be found here.

The required amount of cell culture is taken and centrifuged at 3310 g for 10 minutes.
When the centrifuge is ready, the supernatant is partially removed, and the pellet is resuspended in BHI medium. Note that each culture is resuspended in the appropriate medium.
Set up mCherry dilution series, a dilution of 1:1000 to 1:2000 is recommended. The mCherry is diluted with BHI medium.

To exclude background signal, all media used for cultivation should also be measured.

Before pipetting the samples into the 96-well plate, it is useful to note a scheme in which arrangement the samples should be pipetted into the well plate.

In our Basic Protocols you can find the execution of a spectrum measurement using the example of mCherry.

Detection of Protein Production

Immunoblots

Depending on which constructs were transfected into the Leishmania, the protein of interest will be detectable either in the supernatant or in the lysate. In our case, we have constructs whose protein is directed to be transported out of the cells into the supernatant and constructs where the protein remains in the cell after production. Therefore, the constructs to be analysed must be prepared for an analysis of intracellular expression as well as samples for an analysis of secretory protein expression.

Materials needed

Transfected leishmania cultures are needed in which the production of the protein of interest should be detected. It is beneficial if the cultures have already been induced with tetracycline. It is recommended to induce the cultures with tetracycline two days before use. As negative control the LEXSY starting culture can be used and as positive control e.g., the purified protein against which you want to do your antibody staining.

Further materials

TCA
80% acetone
laemmli (1x)
2-mercapthoethanol

Preparation of the Samples

First you need to count the cells in a counting chamber under the microscope and calculate the required cell culture amount with a cell density of 10⁸ cells/ml. How to calculate the cell culture amount can be found under “Preparing Cultures for Experiments”.

Important: For the sample preparation you must work under the sterile bench.

A. Sample preparation for intracellular expression analysis

The required amount of cell culture is taken and centrifuged at 3310 g for 5 minutes. If an eppendorf tube is not sufficient for the amount of cell culture and a falcon should be used, it is recommended to centrifuge the cell culture a little longer (10-20 minutes).
The supernatant is completely removed.
Resuspend the pellet in 100 µl loading buffer. It is recommended to add 2-mercapthoethanol to the loading buffer.
The samples are placed in the heating block at 97°C for 10 minutes: due to the 2-mercapthoethanol the heating block should be under the fume hood.
If the samples are not loaded onto a gel on the same day, they can be stored at -20°C.

B. Sample preparation for the analysis of secretory protein expression

The required amount of cell culture is taken and centrifuged at 3310 g for 10 - 30 minutes.
The supernatant is removed and transferred to a new eppendorf tube or a falcon.
Important: From now on, work is continued with the supernatant.
From here on, it is no longer necessary to work under the sterile bench. For the following TCA precipitation, however, work should be performed under a fume hood with gown.
For the TCA precipitation, the required amount of TCA is calculated for the individual constructs and the TCA is pipetted onto the samples.
Keep the samples on ice for 30 minutes.
Centrifuge the samples at 15000 g for 15 minutes at 4°C.
Remove the supernatant.
Resuspend the pellet with 1 ml of 80% acetone. It is recommended to wash the pellet 2-3 times with acetone.
Resuspend the pellet in 80 µl loading buffer. Again, 2-mercapthoethanol should be added to the loading buffer.
The samples are placed in the heating block at 97°C for 10 minutes. Because of the 2-mercapthoethanol, the heating block should be under the fume hood.
If the samples are not loaded onto a gel on the same day, they can be stored at -20°C.

Once the samples are prepared, the gel can be loaded for the following immunoblot. The instructions for an immunoblot can be found in our Basic Protocols.

Further Experiments

Growth curve

For the growth curve, any transfected leishmania cultures that have been previously induced with tetracycline are required. Transfected cultures that have not yet been induced can also be used. If this is the case, the growth curve will be more meaningful if the uninduced and induced cultures are from the same colony. The LEXSY original culture can be used as a positive control. Triplicates of each construct are made to use the mean value for evaluation.

Preparation

The growth curve should be performed with a volume of at least 50 ml. For this, before preparing the cells, it must be ensured that the volume of culture present is sufficient. First, the cells must again be counted in a counting chamber under the microscope. Here your culture must be diluted so that you have a cell density of 10⁶ cells/ml on your desired final volume. How to count the cells is described in “Preparing Cultures for Experiments”.

Important: The dilution of the cells should take place under the sterile bench.

The cell culture flasks with the prepared cultures for the growth curve are placed in the incubator at 27°C and must be counted every day from now on until the death phase of the cells has occurred.

Team:TU Kaiserslautern/HowToLeishmania

... HANDLE LEISHMANIA

Introducing Leishmania tarentolae

How to Culture Leishmania

The Raising of the Cultures

The Dilution of the Cultures

Preparing Cultures for Experiments

Induction with Tetracycline

Transfection

Day 1

Day 2

Next step

Measurements in Leishmania tarentolae

Detection of a Successful Transfection

Detection of Protein Production

Further Experiments

Sources