• Beauveria bassiana

Beauveria bassiana

We did some research on Beavearia bassiana fungus because it's an entomopathogenic fungus used during years in agriculture, especially in Canada. [1]

So, we wanted to use this fungus to fight bed bugs which invade our homes. We selected and bought a Beauveria sample.

Growing Beauveria bassiana

First of all, we grew Beauveria bassiana fungus on three different kind of culture medium :

1. LB with an antibiotic Cm50
2. Sabouraud with Cm 50
3. Sabouraud with collagen and Cm 50 [https://2018.igem.org/Team:Aix-Marseille/Protocols]

We made a serial dilution and let the fungus grow at three températures : 25°C, 30°C and 37°C. We obtained a lot of results but the culture medium and the most suitable temperature are the Sabouraud culture medium and 25°C. [2]

Killing insects with Beauveria bassiana

Secondly, we have tested Beauveria Sample on garden bugs with 4g of fungus sample diluted in 40ml of Tween 0,05% to confirmed that it was indeed Beauveria bassiana. We infected them by soaking and observed them for 2 weeks. Unfortunately, results obtained were inconclusive because we had problems keeping our control insect population alive.

However, we did observe mortality, and the infected bugs sprouted fungus a few days after the death of the insects. So, we decided to test our Beauveria Bassiana’s sample on bed bugs in real conditions.

Methionine gamma lyase

Methionine gamma lyase production

Methionine gamma lyase purification

Methionine gamma lyase activity

Mandelate synthase

Mandelate synthase production

Mandelate synthase activity

Chitinase

Chitinase production

To produce chitinase we used our construction BBa_K2718022. An inducible overproduction driven by the pLac promoter, induction experiments using IPTG can show the presence of the protein. We choose to produce chitinase in the E. coli K-12 strain DH5-alpha For this two different clones with chit1 were tested, clone N°10 and N°11.

Figure 1: Figure 1: SDS-PAGE of chitinase produced by E. coli K-12 DH5-alpha.

(put markers lenghts on the image!!!! and indicate chitinase band ) induction conditions etc

As seen in Figure 1, a protein at the expected size (30 kDa) was overproduced in large quantities at 30°C after IPTG induction. The sample not induction with IPTG show only a weak band in the gel at this position. This overproduction is expected to be the chitinase, strangely that clone N°11 did not produce as much chitinase quantity at 16°C and 37°C. We verified that the protein was chitinase using a western blot and an antibody against the C-terminal His-tag we had designed into the protein.

(put image of western and blue of BL21 or not?)

Chitinase production was best after IPTG induction at 30°C. These induction conditions were used to produce a larger quantity of the chitinase for purification and activity tests.

Chitinase purification

The Chitinase designed in biobricks BBa_K2718021 and BBa_K2718022 has a C-terminal polyhistidine-tag which allows it to be easily purified. We purified the Chit1 protein using a His-trap column and an Akta Pure automated chromatography system. This allows a quick and easy purification, the nickel-nitrilotriacetic acid column specifically binds the histidines residues in the tag to the metal ions in the column, and the protein can then be eluted with imidazole.

Figure 2: SDS-PAGE of polyhistidine-tagged chitinase purification produced by E. coli K-12 DH5-alpha, using a HisTrap column.

(put markers lenghts on the image!!!! and indicate chitinase band )

Showed in figure 2, a protein at 30 kDa corresponding at the chitinase is obtained on bacteria lysate, on the non-purified fraction and in the fractions N°6 to N°10. These fractions were pooled and used for activity tests. This result shows that the His tag added to the protein can be used for purification as well as identification using a western blot.

Numerous controls were been done. The chitinase was produced without its peptide signal of secretion, so it will be contained in the cytoplasm, the absence of the band at 30 kDa in the pellet verify that chitinase wasn't contained in the membrane. Bacteria lysate confirmed the presence of chitinase at 30kDa; non-purified migration shows a low quantity of chitinase with all proteins contaminants; the flow through sample didn't contain the protein target demonstrating that all chitinase was fixed on nickel-nitrilotriacetic acid matrix.

The results of chitinase purification prove that chitinase can be purified thanks to its polyhistidine-tag. But purification wasn't optimal and can be improved because a lot of contaminants were isolated with the chitinase. The nickel-nitrilotriacetic acid matrix column used was a bit old, that may have reduced its efficiency. For an optimal separation of chitinase, we should pool eluate fractions containing chitinase and purify until reduce significantly protein contaminants. But due to lack of time, we continued with the 'purified' samples to do the activity tests.

Chitinase activity test

Schales' procedure was used to test the chitinase activity. Schales' procedure is a colorimetric method commonly used for measuring the reducing sugars. Chitin is composed of sugar units of N-acetyl-G-glucosamine. Endochitinase degrades chitin producing shorted N-acetyl-G-glucosamine oligomers each with a reducing end. To monitor chitinase activity, the reducing ends produced after chitin hydrolysis are detected by a reduction in the yellow color of Shales' reagent (measured as the absorbance at xxx nm).

To analyze activity test results, a standard curve was made using the final product of chitinase reaction (N-acetyl-G-glucosamine), and reacting it with Schales' reagent following our protocol.

Figure 3: Standard curve for Schales' test using N-acetyl-glucosamine.

The standard curve shows the absorbance reduction as a function of increasing N-acetyl-G-glucosamine concentration. With this curve, the concentration of final product obtained after chitinase reaction can be determined. Our results show that we can use Schales' procedure to measure N-acetyl-G-glucosamine between 0 and 200 uM.

Figure 4: Table of absorbance measurements of chitinase activity test

Figure 5: Chitinase activity test by Schales' procedure

In figure 4 we see that after 30 minutes of reaction a significant activity was observed. Unfortunately after 1 hour of reaction, the standard deviation is very very high as our replicates were very uneven, we suspect that we made an error in the protocol. Unfortunately, we did not have enough sample or time to repeat the experiment. One probable source of error was the colloidal chitin sample. In this sample, the chitin is not completely dissolved in water but is a suspension with lots of fragments (it is made from the shells of shrimps) this form of the sample is very difficult to pipet accurately. To help with this a centrifugation step was added to the protocol to avoid precipitated chitin scattering light and affecting the results. However, after centrifugation chitin wasn't a hard pellet, it was easily resuspended.

Using our standard curve and our results at 30 minutes, we can conclude that the rate of reaction is about 4 uM of N-acetyl-G-glucosamine / minute.

Figure 6: Table of measurements of controls for chitinase activity test

Figure 7: Controls for chitinase activity test

( comment the results of table and the graph!!!!!)

Trap prototype tests

(put the protocol of trap tests!!!)