iGEM 2018 — Team Aix-Marseille

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# Team:Aix-Marseille/Notebook

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[Protocols](https://2018.igem.org/Team%3AAix-Marseille/Protocols)
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PARTS

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ACKNOWLEDGEMENTS

[Sponsors](https://2018.igem.org/Team%3AAix-Marseille/Sponsors)
[Attributions](https://2018.igem.org/Team%3AAix-Marseille/Attributions)
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# Notebook

[![T--Aix-Marseille--notebook.png](/wiki/images/b/b1/T--Aix-Marseille--notebook.png)](/File%3AT--Aix-Marseille--notebook.png)

WEEK 1

###

## Training

*During the first week, we had several training sessions, under the supervision of Gauthier DANGLA-PELISSIER (Instructor), to get acquainted with the lab work, master everyday protocols (PCR/Colony-PCR, competent cells preparation, transformation, cloning, minipreps, PCR clean-ups...), and establish a classic workflow.*

#### Day 1: Lab setup

We recovered equipment from our home university, cleaned-up the lab, and had a first meeting to establish the project's workflow.

#### Day 2: Preparing [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols") stock and [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols")

We prepared a stock of [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols"). Afterward, we prepared Petri dishes and we [transformed the cells](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") to test their efficiency using a plasmid provided by our instructor.

#### Day 3: Colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") and agarose gel electrophoresis

The next day we had colonies on our Petri dishes and wanted to check if they acquired the transformed plasmid with the right inserted gene. We picked out some supposedly positive colonies and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols"). We then migrated the PCR products on an agarose gel.

#### Day 4: [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols")

We purified the plasmid with the right inserted gene from the confirmed positive colonies using a DNA purification kit.
In a further step, we digested the purified plasmid with respective restriction enzymes to double-check the presence of the inserted gene.

#### Day 5: [Western-blot](/Team%3AAix-Marseille/Protocols#Western_blot "Team:Aix-Marseille/Protocols")

The last day, we checked the induced production rate of the relative protein to the inserted gene (Produced in a preliminary step by our instructor).
WEEK 2

###

*For the whole week, we ran into a lot of problems making [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols"), so we spent a lot of time optimizing the protocol.*

#### Day 1

**Biobricks design**: We designed Hmas, MdlB, MdlC and Hyd1 biobricks.
**Megaprimers design**: We designed megaprimers to add the Tag-his to our biobriks sequences.
**Chitinase biobrick**: We received the chitinase gene sequence from IDT and ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") to amplify it.
**Methionine-γ-lyase biobrick**: We recovered the biobrick from the iGEM 2018 kit.

#### Day 2

**pLacI promoter biobrick**: We recovered the biobrick from the iGEM 2018 kit to assemble it with other biobricks.
**Chitinase biobrick**: We took the PCR product and verified the amplification on an agarose gel. We ran into hybridization problems so we had to determine the optimal hybridization temperature using a gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols").

#### Day 3

**Chitinase biobrick**: We cloned the PCR product in a pSB1C3 plasmid.
**Methionine-γ-lyase biobrick**: We transformed the biobrick (From the iGEM 2018 kit) into BL21 competent strains because we ran into problems making competent DH5α strains .

#### Day 4

**Chitinase biobrick**: We ran another cloning process on the previous [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") product and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Methionine-γ-lyase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on selected colonies. Afterward, we launched starters on the confirmed positive colonies.
**Interlab**: We had a training on the measurement devices and created the respective programs.
**Biobrick design**: We designed a new biobrick (OmpT-AIDAC) a cleavage sequence.
**RFP biobrick**: We recovered the biobrick (From the iGEM 2018 kit) that will be used for white-red screenings.

#### Day 5

**pLacI promoter biobrick**: We transformed the plasmid into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Chitinase biobrick**: We transformed DH5α strains with the plasmid containing the chitinase sequence.
**Interlab**: We recovered the 8 biobricks (From the iGEM 2018 kit) and transformed them into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Methionine-γ-lyase biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and stocked it.
**MdlB biobrick**: We received the biobrick and ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on it.
**RFP biobrick**: We transformed the biobrick into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

WEEK 3

###

#### Day 1

**pLacI promoter biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies.
**RFP biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and launched overnight starters to purify the plasmid later on (No colony-PCR because the colonies are red: RFP expression).
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies.
We amplified the chitinase IDT gene sequence to recover the stock.
**Methionine-γ-lyase biobrick**: We recovered the methionine-gamma-lyase biobrick (from the iGEM 2018 kit) and transformed it into [Team:Aix-Marseille/Protocols#DH5α\_competent\_cells\_preparation|DH5α competent cells]].
**InterLab**: We recovered the 8 [transformations](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") and launched 8 starters in duplicates.

#### Day 2

**pLacI promoter biobrick**: We purified the plasmid with the inserted RFP gene using a DNA purification kits, and restarted because the final DNA concentration was too low (13 ng/µL).
**RFP biobrick**: We purified the inserted RFP gene using DNA purification kits.
**Chitinase biobrick**: We ran a gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") to find the optimal primers hybridization temperature. We then extracted the DNA using a gel extraction kit, digested the gene with respective enzymes, and ran an overnight ligation in a pSB1C3 plasmid at 16°C.
**[DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols")**: We prepared a new [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols") stock and verified their efficiency using an RFP plasmid.
**Methionine-γ-lyase biobrick**: We ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the positive colonies and verified it on an agarose gel.
**InterLab**: We launched the 3 calibration measurements and ran the first cell measurements.

#### Day 3

**pLacI promoter biobrick**: We purified the DNA using a different [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit.
**Chitinase biobrick**: We ran a ligation in pSB1C3 at room temperature.
**[DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols")**: We launched DH5α starters to renew the competent cells stock.
**Methionine-γ-lyase biobrick**: We purified the DNA using the Promega [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit. Afterward, we launched an overnight
[megapriming](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") using specific primers to add a Tag-his to our gene sequence so we can purify the resulting protein later on.
**InterLab**: We analyzed the collected data and launched new starters to run a second measurement test.
**MdlB biobrick**: We ran a ligation in pSB1C3 at room temperature.

#### Day 4

**Methionine-γ-lyase biobrick**: We digested the magapriming product by DpnI (To cut the methylated GATC sites), and transformed it into DH5α competent strains.
**[DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols")**: we renewed our stock and verified their efficiency using the RFP plasmid.
**InterLab**: We ran a second measurement test.
**MdlB biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies.
**Extracurricular**: We made our breaking bugs logo using *E. coli* strains that express the GFP.

#### Day 5

**[DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols")**: We double-checked the competent cells efficiency by transforming the RFP plasmid.
**MdlB biobrick**: We recovered the overnight starters, and purified the DNA using minpreps kits, measured the DNA concentration on the NanoDrop and ran a digestion test.

WEEK 4

###

*We had the same problems making [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols") (contaminations...). We had the help of our instructor who pointed out a lot of flaws with what we were doing in the process.*

#### Day 1

**Chitinase biobrick**: We took stocked strains and launched starters to purify the DNA the next day.
**MdlB biobrick**: We tried cloning the gene sequence into pSB1C3 and transformed the prouct into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols"). Furthermore, we ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the IDT sequence to renew our stock.
**Interlab**: We ran measurments for the third time and launched the CFUs protocol.
**Methionine-γ-lyase biobrick**: We sent the magrapriming product to sequencing to check if we managed to add the Tag-his.

#### Day 2

**Chitinase biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and had 225,87 ng/uL n concentration. Afterward, we sent the product for sequencing, and simultaneously, launched a [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") to add the Tag-his for [purification](/Team%3AAix-Marseille/Protocols#Protein_purification "Team:Aix-Marseille/Protocols") later-on.
**RFP biobrick**: We amplified it to renew our stock for contamination purposes.
**Interlab**: We had a training on how to do dilution series.
**Methionine-γ-lyase biobrick**: The sequencing results were positve: we managed to add the Tag-his to our sequence.
**MdlB biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies.
**Hyd1 biobrick**: We received the gene sequence from IDT, ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on it and stocked it.

#### Day 3

**Chitinase biobrick**: We digested the [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") product by DpnI and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**MdlB biobrick**: We tried cloning the gene sequence, for a second time, into pSB1C3 and transformed the product into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols"). Furthermore, we ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the IDT sequence to renew our stock.
**Interlab**: We launched starters for a second CFUs test.
**Hyd1 biobrick**: We migrated the [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") product on an agarose gel, and did a second [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") after a first failure. After a second failure, we ran a gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") to determine the correct hybridization temperature.

#### Day 4

**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates. No colonies were observable whereas the sequencing results showed positive results (The chitinase sequence was inserted into the pSB1C3 plasmid. We suspected the DH5α cells: turns out they were *Bacillus Subtilis* strains (Smell check). We had to run another [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols").
**MdlB biobrick**: We used the same strains that we used for the chitinase.
**Interlab**: We ran the CFUs test.
**Hyd1 biobrick**: We performed a [PCR clean-up](/Team%3AAix-Marseille/Protocols#Monarch_PCR_and_DNA_cleanup_kit "Team:Aix-Marseille/Protocols") and migrated the PCR product.

#### Day 5

**Chitinase biobrick**: We digested the megapriming product by DpnI and transformed it into new [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Hyd1 biobrick**: We cloned the gene sequence in pSB1C3 and trasnformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

WEEK 5

###

#### Day 1

**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We launched starters from the confirmed positive colonies for the next day.
**Interlab**: We counted the boxes and calculated the CFU/mL. Afterward, we prepared new Petri dishes to run another CFU test.
**Hyd1 biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We had 8 confirmed positive colonies and we launched two starters from two of them for the next day.
**MdlC biobrick**: We received the gene sequence from IDT, then ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") and a gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on it, but we failed.
**Methionine-γ-lyase biobrick**: We ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the pLacI and methionine-gamma-lyase fragments.
**Hmas biobrick**: We received the gene sequence from IDT and ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on it, but failed.

#### Day 2

**Chitinase biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent the product for sequencing.
**Interlab**: We made the dilution series and incubated the plates overnight.
**Hyd1 biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and digested it with specific restriction enzymes. We didn't have conclusive results, so we launched new different starters.
**pLacI promoter biobrick**: We launched a [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") to add the RBS to the promoter.
**Methionine-γ-lyase biobrick**: We assembled the sequence with the pLacI promoter, tried to clone it in pSB1C3, and transformed the product into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

#### Day 3

**MdlB biobrick**: We tried cloning the gene sequence into pSB1C3 and transformed the product into DH5α competent cells.
**Interlab**: We counted the boxes and calculated the CFU/mL. We then elaborated a workflow for the flow cytometry measurements.
**Hyd1 biobrick**: We purified the DNA from a confirmed positive colony using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit.
**pLacI promoter biobrick**: We digested the magapriming product by DpnI (To cut the methylated GATC sites), and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Methionine-γ-lyase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we launched starters on the confirmed positive colonies.

#### Day 4

**MdlB biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We then launched starters from the confirmed positive colonies.
**Hyd1 biobrick**: We sent the miniprep product for sequencing.
**pLacI promoter biobrick**: We launched starters on the confirmed positive colonies for the next day.
**Methionine-γ-lyase biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent the product for sequencing.

#### Day 5

**Chitinase biobrick**: We recovered the sequencing results which were negative.
**pLacI promoter biobrick**: We purified the DNA from a confirmed positive colony using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent the product for sequencing.

WEEK 6

###

#### Day 1

**Methionine-γ-lyase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and launched starters of the supposedly positive colonies with the good gene sequence (Assembled with pLacI).
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We prepared starters from the confirmed positive colonies.
**Interlab**: We prepared 16 starters from our GFP expressing strains for our flow cytometry measurements.

#### Day 2

**MdlB biobrick**: We cloned the gene in pSB1C3 and trasnformed it into DH5α competent cells.
**Methionine-γ-lyase biobrick**: We recovered the sequencing results that were negative (Sequence without pLacI). We to try assembling the pLacI biobrick to are gene sequence a second time.
**Chitinase biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent for sequencing.
**Hyd1 biobrick**: We recovered the sequencing results that were positive.
**Interlab**: We recovered our starters and took the fluorescence measurements on the TECAN and BIO-RAD S3e Cell Sorter flow cytometer.
***Beauveria bassiana*** **growth**: We prepared a specific [medium](/Team%3AAix-Marseille/Protocols#Sabouraud_medium_preperation "Team:Aix-Marseille/Protocols") (Sabouraud) for the fungus so can start the growth process. We had liquid sabouraud stock and sabouraud, agar, and chloramphenicol Petri dishes.

#### Day 3

**MdlB biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We had non-conclusive results.
**Hmas biobrick**: We cloned the gene sequence in pSB1C3 and transformed the product in DH5α competent cells.
**Chitinase biobrick**: We recovered the sequencing results that were negative (No Tag-his). We had to make another [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") to try to add the tag-his on our gene sequencing with minor changes to the protocol.
**Interlab**: We retrieved and analyzed our measurements data.
***Beauveria bassiana*** **growth**: We took aliquots and measured the OD600 to check-out the growth rate. Furthermore, we counted the cells on a Malassez cell.
**Hmas biobrick**: We cloned the gene sequence in pSB1C3 and transformed into DH5α competent cells.

#### Day 4

**Hmas biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepare starters from the confirmed positive colonies.
**Chitinase biobrick**: We digested the megapriming product with DpnI and transformed it into DH5α competent cells.
**Hmas biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We had no positive colonies.
***Beauveria bassiana*** **growth**: We took aliquots and measured the OD600 to check-out the growth rate. Furthermore, we counted the cells on a Malassez cell.

#### Day 5

**Hmas biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and ran a digestion test to double-check if we had the gene inserted.
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates where we had no colonies. Another [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") attent was launched.
***Beauveria bassiana*** **growth**: We took aliquots and measured the OD600 to check-out the growth rate. Furthermore, we counted the cells on a Malassez cell.

WEEK 7

###

#### Day 1

**MdlB biobrick**: We tried optimizing the [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") protocol using different mixes because we were unable to amplify the gene properly. Nonetheless, we failed.
**Lipase biobrick**: Because IDT couldn't provide us with the gene sequence, we tried amplifying it from wild-type *Beauveria bassiana* strains.
**Methionie-γ-lyase biobrick**: We recovered the sequencing results. They were negative, the pLacI assembly to the gene sequence failed.
**Chitinase biobrick**: We digested the megapriming product with DnpI and transformed it in [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**pLacI promoter biobrick**: We launched a [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") to add the RBS to the sequence.
***Beauveria bassiana*** **growth**: We made dilution series on previously launched starters (At different temperatures 25°C, 30°C, and 37°C) to check out the growth rate of the fungus and calculate the CFU/mL.

#### Day 2

**MdlC biobrick**: We ran a gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") using the Phusion-HF DNA polymerase. We had non-conclusive positive results.
**Methionie-γ-lyase biobrick**: We tried assembling the to genes while cloning them (We digested pLacI with SpeI and PstI, and digested The tagged methionine-gamma-lyase sequence with XbaI and PstI.
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates but had no colonies. Afterward, we tried transforming the megapriming product for the second time and added an RFP control.
**pLacI promoter biobrick**: We digested the megapriming product with Dpn1 and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Hmas biobrick**: We purified the DNA from an overnight culture, ran a digestion test and sent for sequencing.
***Beauveria bassiana*** **growth**: We counted the cells using a Malassez cell.

#### Day 3

**MdlC biobrick**: We ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the gene sequence and had positive results. Afterward, we purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and had 70 ng/uL in concentration.
**MdlB biobrick**: We ran another gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") using the Phusion-HF DNA polymerase, and we increased the cycles' number. We had negative results.
**Methionie-γ-lyase biobrick**: We ran the ligation process and transformed the product in [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we launched starters on the confirmed positive colonies.
**pLacI promoter biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we launched starters on the confirmed positive colonies.
**Hmas biobrick**: We recovered the sequencing results that were negative.
***Beauveria bassiana*** **growth**: After 2 days of growth, we found out that the dilution series product are yeast.

#### Day 4

**MdlC biobrick**: We digested the sequence with EcoRI and PstI to clone it in pSB1C3 the next day.
**Methionie-γ-lyase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we launched starters on the confirmed positive colonies.
**Chitinase biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent for sequencing.
**pLacI promoter biobrick**: We purified the DNA using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent for sequencing.
***Beauveria bassiana*** **growth**: We spent time debbugging the problem and tried to find a solution to eliminate the yeats.

#### Day 5

**MdlC biobrick**: We cloned the gene sequence in pSB1C3 and transformed it into competent cells.
**MdlB biobrick**: We ran another [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the gene sequence, but increased the matrix volume in the [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") mix. We had conclusive and positive results.
**Methionie-γ-lyase biobrick**: We purified the plasmid using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent it for sequencing. We then digested the sequence with EcoRI and PstI to clone it in pSB1C3.
***Beauveria bassiana*** **growth**: We recovered the plates growing at 25°C, where we observed preliminary filamentous colonies.

WEEK 8

###

#### Day 1

**MdlC biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates where we had no colonies. We ran another digestion test on the MdlC gene sequence for cloning.
**HmaS biobrick**: We digested the gene sequence with EcoRI and PstI for cloning in pSB1C3. We had the right band at the right size when migrated on an agarose gel.
**Chitinase biobrick**: We ran a gradient-[megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") test using AS-25 chitinase's primers.
**Methionine-γ-lyase biobrick**: We recovered the sequencing positive results. We managed to add the pLacI promoter to the gene sequence. We then digested our DNA with EcoRI an PstI to clone it in the pSB1C3 plasmid.

#### Day 2

**MdlC biobrick**: We cloned the digested gene sequence in a pSB1C3 plasmid and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
We then ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") to renew the DNA stock.
**MdlB biobrick**: We ran gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") using a Phusion DNA polymerase to determine the optimal primers hybridization temperature.
**HmaS biobrick**: We cloned the digested gene sequence in pSB1C3 and transformed into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols"). Afterward, we ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") to renew our DNA stocks but failed.
**Chitinase biobrick**: We digested the megrapriming product by DpnI and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Methionine-γ-lyase biobrick**: We cloned our digested gene sequence in pSB1C3 and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

#### Day 3

**MdlC biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We then prepared starters from the confirmed positive colonies for plasmid purification.
**MdlB biobrick**: We [cleaned-up](/Team%3AAix-Marseille/Protocols#Monarch_PCR_and_DNA_cleanup_kit "Team:Aix-Marseille/Protocols") the gradient-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") product and digested the target plasmid. We had negative results when migrated on an agarose gel.
**HmaS biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepared starters on the confirmed positive colonies for plasmid purification.
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. We then prepared starters from the confirmed positive colonies for plasmid purification.
**Methionine-γ-lyase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepared starters from the confirmed positive colonies for plasmid purification.
**Constructs for** ***Beauveria bassiana***: We searched for plasmids to transform our fungus. We found pEG202 and pJG4-5 (with prokaryotic and eukaryotic replication origins). Afterward, we trasnformed them into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
We designed the *bar* phosphinothricin resistance gene, a biobrick for fungus' growth selection.

#### Day 4

**MdlC biobrick**: We purified the plasmid using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit an sent samples for sequencing.
**HmaS biobrick**: We purified the plasmid and sent samples for sequencing.
**Methionine-γ-lyase biobrick**: We purified the DNA, transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols"), and sent samples for sequencing.
**Constructs for** ***Beauveria bassiana***: We recovered the trandformation plates and prepared starters for plasmid purification.
We recovered three biobricks from the iGEM 2018 kit: GPD eukaryotic promoter, eukaryotic IRES RBS, eukaryotic terminator. Afterward, we transformed them into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

#### Day 5

**Chitinase biobrick**: We purified the plasmid using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit an sent samples for sequencing.
**Methionine-γ-lyase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies.
**Constructs for** ***Beauveria bassiana***: We purified the plasmids using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit. To double-check the plasmids we digested pEG202 with NgoMIV and XbaI and pJG4-5 with AgeI and SpeI.
We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") for double-checking.

WEEK 9

###

#### Day 1

**MdlC biobrick**: We faced a lot of problems with the amplification from the IDT stock. We spent the week debugging and optimizing our protocol.
**HmaS biobrick**: We recovered the sequencing results. We were able to clone the gene. Afterward, we cloned it in another plasmid to add the pLacI promoter. We transformed the product into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
**Chitinase biobrick**: We ran another an exponential [megapriminging](/Team%3AAix-Marseille/Protocols#Exponential_megapriming_PCR_.28EMP.29 "Team:Aix-Marseille/Protocols") PCR to add a tag-his to our gene sequence. Afterward, we digested the [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") product we DpnI and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").
***Beauveria bassiana*** **growth**: We prepared [competent blastospores](/Team%3AAix-Marseille/Protocols#Beauveria_bassiana_competent_blastospores "Team:Aix-Marseille/Protocols") for future use, and stocked aliquotes at -80°C.

#### Day 2

**HmaS biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepared starters from the confirmed positive colonies.
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepared starters from the confirmed positive colonies.

#### Day 3

**HmaS biobrick**: We purified the DNA plasmid using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit and sent samples for sequencing.
**Chitinase biobrick**: We purified the DNA plasmid using a [DNA purification miniprep](/Team%3AAix-Marseille/Protocols#Monarch_miniprep_kit "Team:Aix-Marseille/Protocols") kit. Firstly, we digested the plasmid with XbaI and PstI to clone the gene in a plasmid with pLacI and RBS. Secondly, we digested the plasmid with NgoI and PstI to clone the gene in another plasmid with pLacI, RBS, and a signal peptide.

#### Day 4

**HmaS biobrick**: We recovered the sequencing results. We were able to clone the gene. Afterward, we cloned it in a pSB1C3 plasmid.
**Chitinase biobrick**: We [cleaned-up](/Team%3AAix-Marseille/Protocols#Monarch_PCR_and_DNA_cleanup_kit "Team:Aix-Marseille/Protocols") the digestion products using the Machinery-Nagel kit. Afterward, we cloned the digested genes in respective plasmids and trasnformed the products into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

#### Day 5

**MdlB biobrick**: We received the sequence from IDT with an added tag-his. Then, we ran a [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on it to amplify the gene.
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepared starters from the confirmed positive colonies.

WEEK 10

###

#### Day 1

**MdlC biobrick** and **MdlB biobrick**: For the whole week, we spent a lot of time optimizing the [PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") protocol (Double checking primer annealing, modfying the PCR program...).
**HmaS biobrick**: We transformed the pSB1C3 plasmid in BL21 strains to start the protein production using the following protocol.
**Chitinase biobrick**: We cloned the gene sequence in pLacI pSB1C3 plasmid and transformed into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

#### Day 2

**MdlC biobrick**: After multiple clonings and [transformations](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols"), we were finally able to send purified plasmid samples for sequencing.
**MdlB biobrick**: We kept optimizing portocols.
**HmaS biobrick**: We prepared starters according to the protocol. After the induced production of the protein, we ran a several western blots to check the production rates.
**Chitinase biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates an ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepared starters from the confirmed positive colonies.

#### Day 3

**Chitinase biobrick**: We purified the plasmid and sent samples for sequencing.

#### Day 4

**Chitinase biobrick**: Werecovered the sequecning results. We have a full sequence.

WEEK 11

###

#### Day 1

**Chitinase biobrick**: We started the protein production using the following protocol. (It took 3 days).
**Methionine-γ-lyase biobrick**: We purified the produced protein.
**HmaS biobrick**: We calibrated the [HPLC](/Team%3AAix-Marseille/Protocols#HPLC_.28High_Performance_Liquid_Chromatography.29_test_on_... "Team:Aix-Marseille/Protocols") system in preparations to measurements to test the protein functionality.
**MdlB biobrick**: We recovered isolated colonies and ran an optimized colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols"). Afterward, we prepared starters from the positive colonies.
**MdlC biobrick**: We recovered the sequencing results. We had negatives ones, so we started over trying to optimize the protocols.

#### Day 2

**Methionine-γ-lyase biobrick**: We stored the purified samples at -20°C. Afterward, we created a standard curve to the test the functionality of the protein.
**HmaS biobrick**: We prepared starters to start the HPLC measurements.

[![T--Aix-Marseille--Hplc s mandelate rate evolution.png](/wiki/images/f/fb/T--Aix-Marseille--Hplc_s_mandelate_rate_evolution.png)](/File%3AT--Aix-Marseille--Hplc_s_mandelate_rate_evolution.png)

[![T--Aix-Marseille--Hplc phenylpyruvic acid rate evolution.png](/wiki/images/3/33/T--Aix-Marseille--Hplc_phenylpyruvic_acid_rate_evolution.png)](/File%3AT--Aix-Marseille--Hplc_phenylpyruvic_acid_rate_evolution.png)

You can see how these results have been exploited in the [Experiment](https://2018.igem.org/Team%3AAix-Marseille/Experiments) tab.

**MdlB biobrick**: We purified the DNA and sent samples for sequencing.
**MdlC biobrick**: We digested the gene sequence with respective enzymes, cloned it in a pSB1C3 plasmid, and transformed it into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

#### Day 3

**MdlC biobrick**: We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates and ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies. Afterward, we prepared starters from the confirmed positive-colonies.

#### Day 4

**Chitinase biobrick**: After testing out different production conditions in DH5α strains, we stuck with the most optimal one to get to purify large quantities of the wanted protein.
**MdlC biobrick**: We purified the DNA and sent samples for sequencing.

#### Day 5

**Chitinase biobrick**: After testing out different production conditions in BL21 strains, we stuck with the most optimal one to get to purify large quantities of the wanted protein.

WEEK 12

###

#### Day 1

**Methionine-γ-lyase biobrick :** We [tested](/Team%3AAix-Marseille/Protocols#Methionine-y-lyase_activity_test_using_DTNB_protocol "Team:Aix-Marseille/Protocols") with DTNB (5,5′-Dithiobis2-nitrobenzoic acid) on purified protein and absorbance measurements on the TECAN were performed.

**MdlC biobrick :** We digested the gene sequence with the respective enzymes, cloned it into a pSB1C3 plasmid and extracted it from the gel.

**Chitinase biobrick:** We purified and identified large quantities of the wanted protein.

#### Day 2

**Methionine-γ-lyase biobrick :** We breaked cells with emulsiflex and made activity test on purified protein in triplicate with absorbance measurement on the TECAN.

**MdlC biobrick :** We transformed MdlC in pSB1C3 plasmid.

**MdlB biobrick:** We cloned it in a pSB1C3 plasmid, and transformed it in [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

**Chitinase biobrick:** We prepared activity test solutions (réaction buffer) and discolored gels of the day before.

**Promotor-RBS-peptide signal sequence ompA biobrick :** We cloned RBS promotor and signal sequence in pSB1C3 plasmid and transformed them into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

**Bar biobrick :** We cloned RBS promotor and signal sequence in PSB1C3 plasmid and transformed them into [DH5α competent cells](/Team%3AAix-Marseille/Protocols#DH5.CE.B1_competent_cells_preparation "Team:Aix-Marseille/Protocols").

#### Day 3

**Methionine-γ-lyase biobrick :** We made again activity test in triplicate with absorbance measurement on the TECAN. We start the analyse results.

**MdlC biobrick :** We ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") with Slic1 and 4 on the supposedly positive colonies. Afterward, we prepared starters from the confirmed positive colonies.

**MdlB biobrick:** We ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") with Slic 1 and 4 but we had negatives ones, so we started over trying to optimize the protocols.

**Chitinase biobrick :** We prepared all activity test solutions.

**Promotor-RBS-peptide signal sequence ompA biobrick :** We ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") but we had negatives ones, so we started over trying to optimize the protocols.

**Bar biobrick :** We ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") but we had negatives ones, so we started over trying to optimize the protocols.

#### Day 4

**Methionine-γ-lyase biobrick :** On cell culture of Methionine-γ-lyase needed for the [activity test](/Team%3AAix-Marseille/Protocols#Methionine-y-lyase_activity_test_using_DTNB_protocol "Team:Aix-Marseille/Protocols") (DTNB test and MBTH test), we induced at OD 0,7 with [IPTG](/Team%3AAix-Marseille/Protocols#IPTG_induced_protein_production "Team:Aix-Marseille/Protocols") and incubateur during 5h at 30°C. Measurement of OD was made after incubation and we freezed pellets at 20°C. We made then activity test with MTBH (3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate) on purified protein and absorbance measurement was made on the TECAN but results were not good as expected.

**MdlC biobrick :** We recovered the [transformation](/Team%3AAix-Marseille/Protocols#Transformation "Team:Aix-Marseille/Protocols") plates an ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") on the supposedly positive colonies of last week. Afterward, we prepared starters from the confirmed positive colonies.

**MdlB biobrick:** We ran a colony-[PCR](/Team%3AAix-Marseille/Protocols#Polymerase_Chain_Reaction_.28PCR.29 "Team:Aix-Marseille/Protocols") of last week but we had negatives ones, so we started over to optimize the protocols.

**Chitinase biobrick :** We made an etalon range to realised activity test at 30°C during one hour. 4466,58 ug/mL of chitinase (and contaminants) was obtained.

#### Day 5

**Methionine-γ-lyase biobrick :** We made same activity test with MTBH and absorbance measurement was made on the TECAN but results were not good.

**MdlC biobrick :** We purified the plasmid and sent samples for sequencing.

**Chitinase biobrick :** We made activity tests thanks to [Schales'procedure](/Team%3AAix-Marseille/Protocols#Schales.27_test "Team:Aix-Marseille/Protocols"), a colorimetric method for detecting reduced sugars released by chitin hydrolisis by chitinase.

WEEK 13

###

During the last week in the laboratory, we gathered all the results and analyzed them, and prepared our constructs for shipment to the iGEM headquarters. Afterward, we packed away our equipment and cleaned-up our 3 months workspace.


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# Team:Aix-Marseille/Experiments

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# Experiments

## Contents

* [1 *Beauveria bassiana*](#Beauveria_bassiana)
  + [1.1 Growing *Beauveria bassiana*](#Growing_Beauveria_bassiana)
  + [1.2 Killing insects with *Beauveria bassiana*](#Killing_insects_with_Beauveria_bassiana)
* [2 Methionine gamma lyase](#Methionine_gamma_lyase)
  + [2.1 Methionine gamma lyase production](#Methionine_gamma_lyase_production)
  + [2.2 Methionine gamma lyase purification](#Methionine_gamma_lyase_purification)
  + [2.3 Methionine gamma lyase activity](#Methionine_gamma_lyase_activity)
* [3 Mandelate synthase](#Mandelate_synthase)
  + [3.1 Mandelate synthase production](#Mandelate_synthase_production)
  + [3.2 Mandelate synthase purification](#Mandelate_synthase_purification)
  + [3.3 Mandelate synthase activity](#Mandelate_synthase_activity)
* [4 Chitinase](#Chitinase)
  + [4.1 Chitinase production:](#Chitinase_production:)
  + [4.2 Chitinase purification:](#Chitinase_purification:)
  + [4.3 Chitinase activity test:](#Chitinase_activity_test:)
* [5 Trap prototype tests](#Trap_prototype_tests)

# *Beauveria bassiana*

[![T--Aix-Marseille--(Beauveria bassiana).jpg](/wiki/images/0/09/T--Aix-Marseille--%28Beauveria_bassiana%29.jpg)](/File%3AT--Aix-Marseille--%28Beauveria_bassiana%29.jpg)

We did some research on *Beavearia bassiana* fungus because it's an entomopathogenic fungus used during years in agriculture, especially in Canada.
[[1]](https://pdfs.semanticscholar.org/1463/3f328f5c541399802579e20e153012223f25.pdf)

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 [[2]](https://2018.igem.org/Team%3AAix-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. [[3]](https://www.tandfonline.com/doi/abs/10.4489/MYCO.2009.37.3.218#aHR0cHM6Ly93d3cudGFuZGZvbmxpbmUuY29tL2RvaS9wZGYvMTAuNDQ4OS9NWUNPLjIwMDkuMzcuMy4yMTg/bmVlZEFjY2Vzcz10cnVlQEBAMA==)

## Killing insects with *Beauveria bassiana*

[![T--Aix-Marseille--(First test with Beauveria bassiana with garden bugs).png](/wiki/images/7/75/T--Aix-Marseille--%28First_test_with_Beauveria_bassiana_with_garden_bugs%29.png)](/File%3AT--Aix-Marseille--%28First_test_with_Beauveria_bassiana_with_garden_bugs%29.png)

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

During these 3 months in the laboratory we managed to introduce a plasmid construct containing the LacI promoter inducible with IPTG (<bbpart>BBa\_R0011</bbpart>), RBS + methionine-y-lyase (<bbpart>BBa\_K1493300</bbpart>) and his-tag, inserted in a PSB1C3 vector. In order to test the production of the encoded protein, we did a SDS PAGE from several cellular supernatant as well as western blots.
Regarding the his-tag, the latter has been added to methionine-y-lyase (<bbpart>BBa\_K1493300</bbpart>) with the megaprimer PCR method. The part corresponding to methionine-y-lyase + his-tag is <bbpart>BBa\_K2718005</bbpart>. The part that is used for the protein production is as follows <bbpart>BBa\_K2718006</bbpart>.

[![](/wiki/images/6/6b/T--Aix-Marseille--BluegelDH5a7D.png)](/File%3AT--Aix-Marseille--BluegelDH5a7D.png)

Fig 1. SDS-PAGE DH5a, Induced at 30°C with 1000mM IPTG(7D), Induced at 30°C with 500mM IPTG(6D), Induced at 30°C with 250mM IPTG(5D), Induced at 37°C with 1000mM IPTG(4D), Induced at 37°C with 500mM IPTG(3D), Induced at 30°C with 250mM IPTG(2D), No-induced (1D)

.

To confirm the bands identified on SDS-PAGE corresponds to methionine-y-lyase, a western blot[[4]](https://2018.igem.org/Team%3AAix-Marseille/Protocols) with anti-his-tag antibodies was realized.

[![](/wiki/images/2/2b/T--Aix-Marseille--WBDH5a.png)](/File%3AT--Aix-Marseille--WBDH5a.png)

Fig 2. SDS-PAGE DH5a, Empty plasmid (Ctrl), No-induced (1D), Induced at 30°C with 250mM IPTG(2D),Induced at 37°C with 500mM IPTG(3D), Induced at 37°C with 1000mM IPTG(4D),Induced at 30°C with 250mM IPTG(5D), Induced at 30°C with 500mM IPTG(6D), Induced at 30°C with 1000mM IPTG(7D), Induced at 16°C with 150mM IPTG(8D), Induced at 16°C with 500mM IPTG(9D), Induced at 16°C with 1000mM IPTG(10D)

[![](/wiki/images/d/d2/T--Aix-Marseille--WBBL21.png--Aix-Marseille--WBBL21.png)](/File%3AT--Aix-Marseille--WBBL21.png--Aix-Marseille--WBBL21.png)

Fig 3. SDS-PAGE DH5a, Empty plasmid (Ctrl), No-induced (1B), Induced at 30°C with 250mM IPTG(2B),Induced at 37°C with 500mM IPTG(3B), Induced at 37°C with 1000mM IPTG(4B),Induced at 30°C with 250mM IPTG(5B), Induced at 30°C with 500mM IPTG(6B), Induced at 30°C with 1000mM IPTG(7B), Induced at 16°C with 150mM IPTG(8B), Induced at 16°C with 500mM IPTG(9B), Induced at 16°C with 1000mM IPTG(10B)

The western blot thus confirmed the band identified on the SDS-PAGE corresponded to the methionine-γ-lyase produced by E.Coli DH5α bacteria and by E.Coli BL21 bacteria.

For this test, it used 2 differents strains of E. coli Dh5 alpha which is similar to the strain DH1[[5]](https://biocyc.org/organism-summary?object=ECOL536056), and a BL21 strain[[6]](https://biocyc.org/organism-summary?object=GCF_000022665). First, negatives controls are correct, there is no prodution of methionine-y-lyase with empty plasmid. Then, the bands to approximatively 47kDa[[7]](https://www.uniprot.org/uniprot/Q9SGU9), correspond to methionine-y-lyase molecular weight in both case . Moreover there does not seem to be any significant difference in protein production between the 4 samples when the temperature varies in the DH5 alpha case, whereas this parameter seems to influence production in BL21, since production seems less pronounced at 16 °C. Overall, the production of methionine-y-lyase is lower when the host is a BL21 strain compared to a production in a strain DH5 alpha.
However, the methionine-γ-lyase protein is overproduced. After analyzing these results, the induction condition at 500 mM IPTG at 30 ° C is chosen for the rest of the experiments. This condition is the best in yield / time.

## Methionine gamma lyase purification

After having realized the methionine-γ-lyase production tests, the purification of the latter is realized in order to obtain pure protein fractions. For this, the E. coli DH5 alpha cell lysates transformed with the construct [(BBa\_K2718006)](http://parts.igem.org/Part%3ABBa_K2718006) are purified. Purification of the protein is realized with pure AKTA pure® using a Ni Sepharose 6 column (HisTrap FF[[8]](https://www.gelifesciences.com/en/lt/shop/chromatography/resins/affinity-tagged-protein/histrap-ff-p-00251)). Akta Pure is a automated chromatography system for quick and easy purification.
The fusion of the histine tag with methionine-y-lyase allows to specifically separate the methionine-y-lyase from the total proteins.

[![](/wiki/images/b/b8/T--Aix-Marseille--Shcema_MGL_registre_elution.png)](/File%3AT--Aix-Marseille--Shcema_MGL_registre_elution.png)

Fig 4. Chromatogram of the purification of methionine-y-lyase-histag

In figure 4, there are 2 elution peaks. The peaks are not correctly separed and MGL is eluted on second peak, so the first peak samples contain contaminants. To confirm that the elution peak corresponds to the methionine-γ-lyase, an SDS-PAGE of the fractions corresponding to the elution peak as well as the non-retention is realized (fig.5).The deposit on the SDS-PAGE of the no-retained fraction allows to check if our has been retained on the column [[9]](https://2018.igem.org/Team%3AAix-Marseille/Protocols).

[![](/wiki/images/d/dc/T--Aix-Marseille--HmaS_bleu_purif_registry.png)](/File%3AT--Aix-Marseille--HmaS_bleu_purif_registry.png)

Fig 5. SDS PAGE after purification, NR = non-restraint, 1, 8 and 10-15 correspond to the eluted fractions

After SDS-PAGE visualization (fig. 5), the histidine tag allow to purify the methionine-γ-lyase from the total proteins. An intense band is observable approximately 47 kDa corresponding to methionine-γ-lyase. However, the 47kDa band is observable in the no-restrained, it means that there was too much protein and the used column saturated with our sample. To have a perfect purification of the methionine-y-lyase, it is possible to realize an exclusion chromatography. After purification, a pool of fractions 11 to 26 is made. A volume of 15 ml of methionine-γ-lyase at 2.7 mg / ml is obtained. So for 1l of cell culture 40mg of protein is obtained.
Now that the protein is purified, an activity test is done to check if the methionine is bioactive.

## Methionine gamma lyase activity

To characterize the activity of methionine-γ-lyase, activity tests using DTNB is used. DTNB (5,5′-Dithiobis 2-nitrobenzoic acid) reacts with thiol group so for methionine-y-lyase, DTNB reacts with thiol group of methanethiol.

To realize a methionine-γ-lyase activity test, we were interested in the substrates as well as in the products of methionine-γ-lyase.
We have seen from the enzymatic reaction of methionine that the main subtrate was L-methionine, the products were methanethiol which by oxidation was transformed into DMDS / DMTS and 2-oxobutanoate (see [reaction](http://parts.igem.org/Part%3ABBa_K2718006#Protein_activity)).

By dosing of methanethiol by DTNB thus allows to determinate the activity of methionine-γ-lyase. The activity of methionine-y-lyase can be determined by dosing the second product of the reaction, 2-oxobutanoate, using the MBTH (3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate). The MBTH reacts with alpha-keto of 2-oxobutanoate.

[![](/wiki/images/3/39/T--Aix-Marseille--Shcema_MGL_activity_test_1.png)](/File%3AT--Aix-Marseille--Shcema_MGL_activity_test_1.png)

Fig 6.Results of activity test of methionine-y-lyase using DTNB. Both conditions are : DTNB only and protein + DTNB

In Figure 6, we observe that when comparing condition 1 (L-methionine alone without protein) and condition 2 (L-methionine + methionine-y-lyase), there is a strong increase in absorbance by a factor 12. Condition 1 corresponds to a negative control.

[![](/wiki/images/9/95/T--Aix-Marseille--Shcema_MGL_activity_test_2.png)](/File%3AT--Aix-Marseille--Shcema_MGL_activity_test_2.png)

Fig 7. Results of activity test of methionine-y-lyase using DTNB. Both conditions are : Protein only and protein + DTNB

.

In Figure 7, we observe that when comparing condition 1 (protein alone without the substrate: L-methionine) and condition 2 (L-methionine + methionine-y-lyase), there is a strong increase in the absorbance aproximately of a factor 12. Condition 1 corresponds to second negative control as in Figure 6.

These large increases in the absorbance between the two conditions (FIG. 6 and FIG. 7) biologically indicate that methionine-γ-lyase transforms L-methionine into methanethiol. We can therefore deduce that methionine-y-lyase is bioactive.

# Mandelate synthase

## Mandelate synthase production

During the 3 months in the laboratory we managed to introduce a plasmid construct containing the IPTG promoter, the RBS and the hmaS gene, inserted in a PSB1C3 vector. In order to test the production of the encoded protein, we did a SDS PAGE from several cellular fractions [[10]](https://2018.igem.org/Team%3AAix-Marseille/Protocols).

[![T--Aix-Marseille--hmaS production - DH5 alpha E.coli strain.png](/wiki/images/3/3a/T--Aix-Marseille--hmaS_production_-_DH5_alpha_E.coli_strain.png)](/File%3AT--Aix-Marseille--hmaS_production_-_DH5_alpha_E.coli_strain.png)

Figure 1 SDS-PAGE of HmaS production with induction (2, 3 and 4), without induction (1,NI) and with empty plasmid (5), into a DH5 alpha *E.coli* strain.

[![T--Aix-Marseille--hmaS production - BL21 E.coli strain.png](/wiki/images/0/0a/T--Aix-Marseille--hmaS_production_-_BL21_E.coli_strain.png)](/File%3AT--Aix-Marseille--hmaS_production_-_BL21_E.coli_strain.png)

Figure 2 SDS-PAGE of HmaS production with induction (2, 3 and 4), without induction (1,NI) and with empty plasmid (5), into a BL21 *E.coli* strain.

For this test we used 2 different strains of *E. coli* Dh5 alpha which is similar to the strain DH1 [[11]](https://biocyc.org/organism-summary?object=ECOL536056), and a BL21 strain [[12]](https://biocyc.org/organism-summary?object=GCF_000022665).
First, negative control that we made are as expected, there is no prodution of HmaS with an empty plasmid.
Then we can see bands at approximatively 37kDa, that correspond to HmaS molecular weight in both cases [[13]](https://www.uniprot.org/uniprot/G4V4S7). Moreover, there does not seem to be any significant difference in protein production between the 4 samples when the temperature varies, in the case of DH5 alpha, whereas this parameter seems to influence production in BL21, since production seems less successful at 16 °C. Overall, the production of HmaS appears to be lower when the host is a BL21 strain compared to a production in a strain DH5 alpha. However what we can say now is that we found what seems to be an HmaS overproduction.

## Mandelate synthase purification

After HmaS production tests, and in order to obtain pure fractions of the protein, we tried to purify the protein from the cell lysate of a strain of DH5 alpha transformed with our construct[BBa\_K2718011](http://parts.igem.org/Part%3ABBa_K2718011).
For this we did a purification on AKTA with a Resource Q anion exchange column[[14]](https://2018.igem.org/Team%3AAix-Marseille/Protocols).

[![T--Aix-Marseille--hmaS purification - AKTA.png](/wiki/images/d/d1/T--Aix-Marseille--hmaS_purification_-_AKTA.png)](/File%3AT--Aix-Marseille--hmaS_purification_-_AKTA.png)

Figure 3 Elution of HmaS after purification by ion exchange column performed by Akta

During the first chromatography we can see 3 peaks in the elution profile which appear between 85 and 125 ml of solution B.
One of them seems to be our protein however results are not perfect, because ion exchange chromatography is not a selective affinity technique for protein isolation.
That is why we decided to do SDS-PAGE on the elution fractions to see if the protein is present (see figures 4a and 4b)[[15]](https://2018.igem.org/Team%3AAix-Marseille/Protocols)

[![T--Aix-Marseille--Post purification SDS PAGE 1.png](/wiki/images/5/55/T--Aix-Marseille--Post_purification_SDS_PAGE_1.png)](/File%3AT--Aix-Marseille--Post_purification_SDS_PAGE_1.png)

Figure 4a SDS-PAGE of eluate after purifiction L. Ladder , 1'Bacterial lysate ; 2'pellet of bacteria lysate ; 3' non purified fraction ; 4' flow through during protein injection ; 5' flow through during wash before elution ; 3,8,9 and 10 elution samples

[![T--Aix-Marseille--Post purification SDS PAGE 2.png](/wiki/images/5/55/T--Aix-Marseille--Post_purification_SDS_PAGE_2.png)](/File%3AT--Aix-Marseille--Post_purification_SDS_PAGE_2.png)

Figure 4b SDS-PAGE of eluate after purifiction L. Ladder 11 to 24 elution sample

The very intense spots, at approxomatively 37kDa, can be seen, which probably corresponds to HmaS.
Nevertheless, the samples were not pure, others proteins were mixed with HmaS.
However, what we can say is that HmaS is on fractions 8 to 17, which correspond to the second peaks on figure 3.
In conclusion, we partly purifed HmaS. Moreover, we can improve this purification, by using other methods like size eclusion chromatography.

## Mandelate synthase activity

Then we tested the activity of our [BBa\_K2718011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718011) biobrick.
In order to test the mandelate synthase activity in our *E.coli strain*, we made an HPLC analysis of cell supernatents over 7h of cell culture as described in our [protocols tab](https://2018.igem.org/Team%3AAix-Marseille/Protocols).
The elution profils have been analysed and were compiled into several figures that can be found in the [Notebook](https://2018.igem.org/Team%3AAix-Marseille/Notebook). All these data have been converted in order to substract the control values (DH5 alpha) and we have concentrated on the first 3h. During the exponential phase of growth we saw a significant production. The resultant graphs are shown here.

[![T--Aix-Marseille--Hplc phenylpyruvic acid rate evolution new.png](/wiki/images/f/fc/T--Aix-Marseille--Hplc_phenylpyruvic_acid_rate_evolution_new.png)](/File%3AT--Aix-Marseille--Hplc_phenylpyruvic_acid_rate_evolution_new.png)

[![T--Aix-Marseille--Hplc s mandelate rate evolution new.png](/wiki/images/2/25/T--Aix-Marseille--Hplc_s_mandelate_rate_evolution_new.png)](/File%3AT--Aix-Marseille--Hplc_s_mandelate_rate_evolution_new.png)

Fig 5e. Hplc test results: (S)-mandelate rate evolution

Fig 5f. Hplc test results: Phenylpyruvic acid rate evolution

The first thing that can be noticed is the global decrease in phenylpyruvic acid and increase in (S) mandelate concentrations.
There is some mandelate production in the control strain possibly due to non-enzymatic degradation of the phenyl-pyruvate.
We can conclude that there seems to be a (S) mandelate synthase activity, as the (S) mandelate rate are highter in our transformed strain than the *E.coli* strain with empty plasmid (in particular with the psb1c3 plamsid).
However, we have not yet confirmed these results.
In addition, the HPLC peak separation is not perfect, so a modification of the gradient parameters between solution A and B, as well as elution time, might improve the results.

# Chitinase

## Chitinase production:

We managed to produce chitinase thanks to our construction [BBa\_K2718022](http://parts.igem.org/Part%3ABBa_K2718022).
An inducible overproduction driven by pLac promoter was used to study chitinase production, induction experiments using IPTG showed the presence of the protein. We choose to produce chitinase by E. coli K-12 DH5-alpha strain.
For this two strains two differents clones N°10 and N°11.

[![T--Aix-Marseille--ChitinaseResults1.jpg](/wiki/images/c/cf/T--Aix-Marseille--ChitinaseResults1.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults1.jpg)

Figure 1: SDS-PAGE of chitinase produced by E. coli K-12 DH5-alpha (Ladder: 250kD, 130kD, 100kD, 70kD, 55kD, 35kD, 25kD, 15kD, 10kD).

As seen in Figure 1, a protein at the expected length (30 kDa) was overproduced at 30°C after IPTG induction in DH5alpha.
The un-induced cells show an low level of protein production.
This overproduction should be the chitinase, but it strange that the clone N°11 wasn't able to produce much protein at 16°C and 37°C. We verified that the protein was chitinase with a western blot.

[![T--Aix-Marseille--ChitinaseResults12.jpg](/wiki/images/e/ec/T--Aix-Marseille--ChitinaseResults12.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults12.jpg)

Figure 2 : Western blot with anti-HIS antibodies revelation

Chitinase production was best after IPTG induction (at 500 mM) at 30°C.
This condition of culture will be used to produce in large quantity chitinase for purification and activity tests.

## Chitinase purification:

Chitinase designed had a polyhistidine-tag which allows it to be specifically separated from the total proteins.
A purification using a His-trap collumn was used to purify Chit1, on an Akta Pure automated chromatography system.
The nickel-nitrilotriacetic acid column matrix allows a specific interaction between histidine residues and the bound metal ions of the nickel-nitrilotriacetic acid.

In figure 3, an elution peak corresponding to the tagged chitinase visible,
but a slight curve at the start of elution peak perhaps indicates a contaminant peak.
There isn't a separation of these two peaks therefore contaminant proteins and protein of interest are eluted togeather in the first samples.
According to the elution profile and SDS-PAGE, chitinase is present in fraction 6 to 9, but these samples weren't completely pure, a lot of nonspecific proteins were recovered.
Another purification is required for better purity, unfortunately we didn't have time to repeat the purification.
But we prove here that chitinase should be purified thank to its polyhistidine tag.

[![T--Aix-Marseille--ChitinaseResults11.jpg](/wiki/images/d/d7/T--Aix-Marseille--ChitinaseResults11.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults11.jpg)

Figure 3: Elution graph from chitinase purification

[![T--Aix-Marseille--ChitinaseResults14.jpg](/wiki/images/9/92/T--Aix-Marseille--ChitinaseResults14.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults14.jpg)

Figure 4: SDS-PAGE of polyhistidine-tagged chitinase purification produced by E. coli K-12 DH5-alpha, using a nickel-nitrilotriacetic acid matrix.

As shown in figure 4, a protein at 30 kDa corresponding at the chitinase is obtained in the bacterial lysate,
in the non-purified fraction and in fractions N°6 to N°10. These fractions were pooled and used for activity tests.
Numerous controls have been done,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; break-though 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 ameliorated because a lot of contaminants are isolated with chitinase. The nickel-nitrilotriacetic acid matrix column used was well-used, that can reduced its efficiency. For an optimal separation of chitinase, we should pool eluted fractions containing chitinase and purify to reduce the protein contaminants. But due to lack of time we continued with this purification to do activity tests.

## Chitinase activity test:

Schales' procedure was used to test the chitinase activity. Schales' procedure is a colorimetric method commoly used for measuring the reducing sugars.
Chitin is composed of sugar units of either N-acetyl-G-glucosamine. Chitinase degrade chitin producing N-acetyl-G-glucosamine. To monitor chitinase activity, the reducing sugars released after chitin hydrolyse are detected by a color diminution of the yellow Shales' reagent. This color diminution is translated by a absorbance diminution.

To analyse activity test results, an etalon curve have been done using the final product of chitinase reaction (N-acetyl-G-glucosamine), to know in which concentration of final product must be obtained after reaction to be detected by the method of Schales.

[![T--Aix-Marseille--ChitinaseResults3.jpg](/wiki/images/3/3b/T--Aix-Marseille--ChitinaseResults3.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults3.jpg)

Figure 5: Etalon curve of N-acetyl-G-glucosamine after Schales's activity test.

A final curve has been obtain showing a absorbance diminution in function of increasing N-acetyl-G-glucosamine concentration.
With this etalon curve, concentration of final product obtain after chitinase reaction can be determined. This results revealed the efficiency of Schales'procedure to detected N-acetyl-G-glucosamine to 0 at 200 uM.

[![T--Aix-Marseille--ChitinaseResults9.jpg](/wiki/images/5/5f/T--Aix-Marseille--ChitinaseResults9.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults9.jpg)

Figure 6: Table of absorbance measurements of chitinase activity test

[![T--Aix-Marseille--ChitinaseResults4.jpg](/wiki/images/b/b1/T--Aix-Marseille--ChitinaseResults4.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults4.jpg)

Figure 7: Chitinase activity test by Schales' procedure

According to figure 7, after 30 minutes of reaction a significant activity was mesured.
But after 1 hour of reaction the standard deviation was unfortunately very large.
One of the difficulties encountered with the assay was the chitin sample. Colloidal chitin is suspended in water, but made from partially hydrolysed shrimps shells and this was difficult to pipet and caused problems with reproducibility.
We added a centrifugation step to the protocol but precipitated chitin sometimes became resuspended and altered the results.
After centrifugation chitin wasn't really pelleted, it was easily removed.
This problem with chitin pelets is the origin of the high standard deviation in the 1hour point.

Thanks to our standard curve, considering the results after 30 minutes, we can conclude that the initial rate of the reaction, our chitinase can released 125 uM of N-acetyl-G-glucosamine. An initial rate of chitin hydrolysis of 4 uM/min.

[![T--Aix-Marseille--ChitinaseResults10.jpg](/wiki/images/a/ad/T--Aix-Marseille--ChitinaseResults10.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults10.jpg)

Figure 8: Table of measurements of controls for chitinase activity test

Controls were realized to be sure that the reduction of absorbance is due to chitinase reaction. A control with Schales'reagent and water show a hight absorbance by the colorimetric reactant, when chitin was added absorbance decreased that was expected because of chitin reducing sugar ends.

[![T--Aix-Marseille--ChitinaseResults5.jpg](/wiki/images/6/6e/T--Aix-Marseille--ChitinaseResults5.jpg)](/File%3AT--Aix-Marseille--ChitinaseResults5.jpg)

Figure 9: Controls for chitinase activity test

# Trap prototype tests

[![T--Aix-Marseille--Traped bed bug new.png](/wiki/images/4/44/T--Aix-Marseille--Traped_bed_bug_new.png)](/File%3AT--Aix-Marseille--Traped_bed_bug_new.png)

We decided to conduct field tests with a trap prototype a few weeks before the Giant Jamboree to test the efficiency of both units: *Beauveria bassiana* combined with adjuvants, and the pheromones efficiency. During one week, we put traps containing our fungus, the pheromones or both of them, in infested places with bed bugs. The protocol is available on our [Protocols](https://2018.igem.org/Team%3AAix-Marseille/Protocols) tab.
The traps observation after recovery and freezing showed us that we caught a bedbug in one of our traps containing the pheromone cocktail and *Beauveria bassiana*. So we can conclude that the bedbugs do visit our traps.

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Aix-Marseille/Results

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[HOME](https://2018.igem.org/Team%3AAix-Marseille)
[TEAM](https://2018.igem.org/Team%3AAix-Marseille/Team)

PROJECT

[Description](https://2018.igem.org/Team%3AAix-Marseille/Description)
[Design](https://2018.igem.org/Team%3AAix-Marseille/Design)
[Experiments](https://2018.igem.org/Team%3AAix-Marseille/Experiments)
[Notebook](https://2018.igem.org/Team%3AAix-Marseille/Notebook)
[Protocols](https://2018.igem.org/Team%3AAix-Marseille/Protocols)
[InterLab](https://2018.igem.org/Team%3AAix-Marseille/InterLab)
[Model](https://2018.igem.org/Team%3AAix-Marseille/Model)
[Results](https://2018.igem.org/Team%3AAix-Marseille/Results)
[Perspectives](https://2018.igem.org/Team%3AAix-Marseille/Perspectives)
[Safety](https://2018.igem.org/Team%3AAix-Marseille/Safety)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AAix-Marseille/Parts)
[Basic Parts](https://2018.igem.org/Team%3AAix-Marseille/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AAix-Marseille/Composite_Part)

HUMAN PRACTICES

[Integrated HP](https://2018.igem.org/Team%3AAix-Marseille/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AAix-Marseille/Public_Engagement)
[Collaborations](https://2018.igem.org/Team%3AAix-Marseille/Collaborations)

ACKNOWLEDGEMENTS

[Sponsors](https://2018.igem.org/Team%3AAix-Marseille/Sponsors)
[Attributions](https://2018.igem.org/Team%3AAix-Marseille/Attributions)
[Crowdfunding](https://2018.igem.org/Team%3AAix-Marseille/Donors)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Aix-Marseille)

# Results

Positive thoughts, positive actions and positive results. As a hard working team, we aimed high this year. We have generated results that met with the bronze, silver and gold medals requiremements. We were able to produce a functional endochitinase (Gold medal criterion), metabolic intermediates from the benzaldehyde/benzylalcohol biosynthesis pathway, and the DMTS/DMDS pheromones (Silver medal criterion) . We have successfully modeled the trap (Gold medal criterion) and tested it in infested premises. Furthermore, we have made collaborations (Silver medal criterion) with the iGEM committee, as well as with private companies. This sets a solid ground for further developments around the project. Last but not least, a nationwide advertisement campaign engaged the public in our adventure. We had a mutual interest: all bed bugs must come to an end.

# Wetlab

## [Endochitinase](https://2018.igem.org/Team%3AAix-Marseille/Experiments)

[![T--Aix-Marseille--(fourmis).jpg](/wiki/images/b/b2/T--Aix-Marseille--%28fourmis%29.jpg)](/File%3AT--Aix-Marseille--%28fourmis%29.jpg)

The endochitinase is a protein naturally produced by *Beauveria bassiana* an enthomopathogenic fungus. We wanted to overproduce it (in *E. coli*) and extract it to enhance the killing efficieny and speed of the fungus propagation.
We succesfully produced, and purified the functional endochitinase ([BBa\_K2718022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718022)).
This part is an improvement of the part [BBa\_K1913000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K1913000) **Gold medal criteria**.

## Pheromone production

The aim was to produce two types of pheromone: DMTS/DMDS and benzaldehyde/benzylalcohol. We did some research on the two molecules to get the biosynthesis pathways to produce them in *E. coli*. First, we needed to add the three enzymes implicated in the biosynthesis of benzaldehyde/benzylalcohol: MdlB, MdlC and Hmas. We successfully cloned the Hmas gene ([BBa\_K2718011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718011)) and produced the equivalent protein. This enabled [the production of the first metabolic intermediate](https://2018.igem.org/Team%3AAix-Marseille/Experiments) (S-Mandelate) of the pathway.
Second, we were able to clone the methionine-γ-lyase gene ([BBa\_K2718006](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718006)) and produce the equivalent protein. This enabled the achievement of a first goal: the production of the DMTS/DMDS pheromone. This part [BBa\_K2718006](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2718006) is our validated part **Silver medal criteria**

## Trap tests

A few weeks before the giant jamboree we have decided to go all the way with the breaking bugs project. We took a preliminary [trap design](https://2018.igem.org/Team%3AAix-Marseille/Design) to the field to test the efficiency of both units: *Beauveria bassiana* combined with the adjuvants, and the pheromones efficiency. In collaboration with local infested residences, we had the chance to demonstrate some functional aspects about the prototype.

## [Model](https://2018.igem.org/Team%3AAix-Marseille/Model)

[![T--Aix-Marseille--run bedbug 1.png](/wiki/images/7/78/T--Aix-Marseille--run_bedbug_1.png)](/File%3AT--Aix-Marseille--run_bedbug_1.png)

We decided to model the deployment of our bed bug trap in a realistic environment, to understand how it was likely to work and what parameters might be important. This task involved a number of different modeling tasks, that posed different problems: modelling the diffusion of pheromones in the room coming both from a natural bed bug nest and the traps that we plan to deploy; modelling the movement of bed bugs influenced both by the pheromone field and their nest; and modeling the fungal epidemic that we plan to induce in the bed bug population. Though the model is complex and includes many parameters, it has already allowed us to draw several conclusions, and as the model is improved and the parameters are refined other conclusions will follow.

For instance, using realistic diffusion parameters it is clear that the pheromone field is relatively rapidly established and so the precise nature and concentration of pheromones is probably not critical. In contrast, the delay between infection and death of bed bugs is critical for ensuring the eradication of the nest. Our modeling thus helps understand critical aspects of the proposed design. This is a **Gold medal criteria**.

## [Interlab](https://2018.igem.org/Team%3AAix-Marseille/Interlab)

The InterLab study has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. GFP was chosen as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein. This year, we helped in answering the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
We participated in this **Bronze medal criteria**

## [Perspectives](https://2018.igem.org/Team%3AAix-Marseille/Perspectives)

The nationwide success of our approach to treat bed bugs has encouraged us to elaborate perspectives for long term development. We are collaborating with multiple potential investors we have met at the [social housing union convention](https://2018.igem.org/Team%3AAix-Marseille/SHUC) and [Rentokil](https://contact.rentokil.fr/fr-deratisation-v3/?utm_term=rentokil&gclid=CjwKCAjwu5veBRBBEiwAFTqDwYY8qKmbHDOWKutmcTcQWmW9qgyysHT0DWQj_fK3BRepTRwrgH8MIBoCjJQQAvD_BwE) for further research and development around our plan.

## [Collaborations](https://2018.igem.org/Team%3AAix-Marseille/Collaborations)

Connecting with other iGEM teams from all around the world was the power engine of our project. We managed to make collaborations on many levels. We helped the Mexican team with their project's advancement, the German team with their awareness campaign and the Chinese team with their international language project. Additionnally, we organized the 2nd edition of the Mediterranean meetup to help the fellow iGEMers prepare for the giant jamboree. This is a **Silver Medal criteria**

## [Human Practices](https://2018.igem.org/Team%3AAix-Marseille/Human_Practices)

The public engagement was the pillar of the breaking bugs project. After a succesfull nationwide adverstisement campaign, we were able to engage the french and international commitees in our plan. We had a solid validation by a massive waves of donations for our crowfunding campaign.
Our human practices and public engagement was very strong, completing a **Silver medal criteria**, and we integrated our human practices work into the project design a **Gold medal criteria**.