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Notebook

Week 1 - February 15th to 21st

2020’s Aix Marseille iGEM Team introduced to the new team the whole iGEM organisation, explained how to choose a relevant project for 2021 iGEM competition. The new team voted for a primary and a secondary secretary and filled out forms. Each member of the team had to choose in which division they wanted to work in: Business / Head-hunter / Stalker / Communication…

A form to choose the project has been created and sent to the team.

Week 2 - February 22nd to 28th

The Each division presented their research and their advancement. The different project’s ideas have been quickly presented. As there are too many ideas, there is a need to reduce the number of ideas for the next meeting. For this purpose, each member of the team had to prepare one slide per topic.

Week 3 - March 1st to 7th

All the project’s ideas have been presented. The sixteen initial topics have been divided into three major sectors: Atmosphere pollution, oceans pollution and energy production. In each theme, two to four prblematics will be presented into more details for a future vote.

Week 4 - March 8th to 18th

All social networks have now their responsible person.

The vote for the project was stopped on the 3rd of March. Four problematics caught the eye: Atmospheric pollution, tiger mosquitoes, fossil resources and microbial fuel cells. A vote has been done during the meeting, and two topics stand out: Tiger mosquitoes and fossil resources. Extensive research will be done on those subjects and will be presented in the next meeting.

Wetlab

Week 5 - March 19th to 28th

After intense brainstorming and exchanges: Tiger Mosquitoes has been chosen as the 2021 topic! Each member of the team will have to work on various aspects of the following problem: How could we kill only tiger mosquitoes infected by an arbovirus. Indeed, nowadays there is no vaccine against arboviral diseases such as Dengue, Zika or Chikungunya, and the only way to prevent infections is to spread pesticides that impact the whole fauna and flora. The team now need to look for an innovative approach, environmentally and ecosystem friendly to drive mosquitos’ death when a virus is detected into it.

On another hand, a first contact with Promega has been done to create a partnership.

Week 6 - March 29th to April 3rd

During this meeting, the whole team worked on the design of the 2021 project. The main idea is to use a bacterium, ideally a natural mosquito’s host, to produce and release or keep into its membrane, a molecule/protein able to recognize any arboviruses present into the mosquito. When an infection is detected through this specific protein, it will induce a signalling pathway which at the end will be able to induce mosquito’s death.

Consequently, questions need to be investigated: Who composed the mosquito's microbiota? Can they be transmitted through generations? Do mosquitoes have a natural immune system against arboviruses? What are the proteins and pathways involved in it? Can we make them produced by the bacteria of interest? What is the composition of arboviruses (genetic, capsid…)? What are the molecules or systems known to kill mosquitoes? How can we induce mosquito’s death through bacteria?

Week 7 - April 4th to 11th

After previous brainstorming, the team has looked for a microorganism which naturally lives in the mosquito’s microbiota to engineer it and used it as an arbovirus detector. As Bacillus thuringiensis has already been studied, the team decided to not focus on this microorganism. Instead, we have found Asaia bacteria, a natural host of mosquito’s gut which can be transmitted horizontally and vertically. Asaia will be the bacteria of interest to engineer a specific molecule onto it, able to recognize viruses and induce a specific response.

Week 8 - April 12th to 18th

The team was able to have more information about arboviruses in general. Arboviruses stand for Arthropods borne viruses meaning that they are transmitted via insects. There are more than five hundred known arboviruses. In France, West Nile Fever, Dengue and Chikungunya are present, however they are more found in tropical areas. Mosquitoes have natural defences to fight against arboviruses via signalling pathways specifically activated during viral infections such as RNAi, JAK-STAT, Toll or IMD pathways. The latter cited pathways will be investigated for next week's meeting in order to find a protein/molecule able to recognize and induce a response against arbovirus.

Week 9 - April 19th to 26th

During this meeting, we have raised the problem of arbovirus detection. We could induce the production of a specific molecule by the bacterium. As said in the previous meeting, mosquitoes have natural protein to fight viral infection. Those protein could be transfected and produced by Asaia by using a plasmid encoding for it. The protein of interest could be excreted, after being produced, through a secretion system. Asaia possess T1SS and T2SS.

However, it seems difficult to transfect a eukaryotic protein into the bacteria and have it produced.

On the other hand, it seems complicated to find a molecule produced by a bacteria able to recognize arbovirus within the mosquito.

Finally, the team has found an interesting gene implicated in induced insect death: tTAV.

Week 10 - April 26th to May 2nd

This week, each part of the design was studied intensively by a group of students.

First, the team tried to find a “natural” way to detect the arbovirus within the mosquito’s gut by playing with the mosquito’s immune system. Indeed, the mosquito possess different molecules groups involved in the activation of the immune system (antibacterial activity, viral elimination, lectin C encoding, plasmodium elimination). The most known are the JAK-SAT pathway, Toll pathway and IMD pathway.

On a second part, we tried to find a signalisation cascade, going from the arbovirus recognition to the production of toxic molecules. We were thinking to use a Two-component system, involved in bacterial Quorum Sensing e.g.. However, the main problem that we encounter for this system is the low specificity from the receptor present in the two-component system. Indeed, as it participates in intra and inter-communication within bacterial community, it could have an impact on neighbouring species.

To find a way to kill the mosquito, the team has looked for previous iGEM’s project working with Asaia genus to have deeper knowledges on how to use this bacterium in the laboratory. Team Lausanne from 2010 have worked with Asaia to fight against Malaria. Indeed, we were able to collect protocols for Asaia culture and competent cells obtention, signal peptide from pelB used by Lausanne Team to secrete the toxin of interest. This will help us for the last part of our design: toxin production to induce mosquito’s death.

Week 11 - May 3rd to 9th

During this week, the business team has worked on funding files to raise funds for the project as the experiments will begin in two months from now.

The Event’s team is looking for a festival, scientific event close to Marseille to register the Aix Marseille team.

Finally, the head-hunter is looking for students with strong computer modelling skills, to help us in the future of our project.

A major step in our project is to find a name to it! Each member of the team has chosen a name and a vote will be conducted in the next few days.

About the scientific and the most important part of the project, the seventh brainstorming was conducted during this meeting.

Which specific molecule, from the virus, could we easily detect?

Arboviruses are protected by a capsid, a membrane (Protein M) and an envelope formed by E proteins. Consequently, the envelope proteins will be our target to detect the virus. E proteins can interact with lectins from the cell membrane. The idea behind that is to use a lectin to detect the arbovirus. ConA, is one of them and can be produced in E. coli. The protein will need to be first produced and then secreted into the extracellular space.

Which secretion system could we use? T1SS, bacterial lysis, or other?

T1SS (PelB) is the only secretion system found in Asaia genus.

How to engineer / construct a system to convey the information from the detection to the gene?

The AHL, a sensor molecule from a two-component system found in bacteria, could be linked to the lectin ConA. When the latter recognizes the virus, the complex will interact with the responsive element to induce gene translation. The problem here is the glycosylation in the target bacteria and the specificity of the AHL system.

After this long meeting, we must focus on:

Alternative to AHL: we need a receptor/ligand with high specificity and already known (that we do not have to engineer).

Secretion system: investigate on pelB and SST1.

Bacterial lysis to induce mosquito’s death: what is the required concentration of tTAV to kill the mosquito? which strong promoter could we induce to induce tTAV production? Which gene to induce bacterial lysis?

Week 12 - May 10th to 16th

First group has worked on the PelB system and SST1. PelB has already been produced by other iGEM’s teams and the SST1 could be easily used to secrete diverse molecules including functionals adhesins. The sequence of pelB has been collected.

Second group focused on the signal translocation to activate tTAV production by using AHL. However, this solution will not be retained because of the 3D conformation of the AHL. Indeed, after the interaction of ConA/Protein E, there will be no conformational changes in the molecule that could enhance a higher specificity for the signal transduction. This section still needs to go under intense research.

If we keep ConA as a detector of arboviruses, we could use it in a dimeric conformation, known to have higher recognition of viral proteins.

To solve the glycosylation problem, a group has found a strain from E. coli able to realize N-glycosylation: CLM37.

Third group has research lysis protein. Holin protein is responsible for bacterial lysis and in our project, could be used to secrete toxin against mosquito within his gut. Holin/endolysin has been found in iGEM’s biobricks and seems to work very efficiently. For the construction, we need an operon with a strong promoter to produce in sufficient amount tTAV and a weak promoter for holin/endolysin production.

Week 13 - May 17th to 23rd

This week was super intense for the business team, they have filled up forms to have different subventions.

Additionally, during previous weeks, we have contacted Litchi company to create our promotion video! Today it is our first meeting with François Reimbold to set up the model of the video and the associated text. We will have meetings every 2 weeks to choose the design, validate the text, choose the voice, and other parameters.

A “postcard project” has been set up with Dusseldorf’s team in which the team has to present the project with a picture linked with it. The postcard will be sent worldwide to other iGEM teams.

For the scientific part, Nicola has studied the construction for ConA production. The best conformation for the ConA protein is the dimeric structure. However, ConA is present under the tetrameric form. Hence, amino acids need to be replaced by others to decrease the interaction and avoid the formation of a tetramer of ConA.

Another team has research for a promoter that could be used to induce bacterial lysis. Either promoter using an alternative sigma (Rpoa, Rpoc, Rpod,) or viral promoters.

Week 14 - May 24th to 30th

10th brainstorming about our project: ARBO-BLOCK!
As usual, we follow the design of our major idea to conduct the research:
ConA could be fused with a molecule able to change its conformation when an interaction with an arbovirus is made. The system will then liberate a domain to be cleaved by a peptidase. The released domain can then enter the cell through a porin to induce the phosphorylation cascade and at the end cell lysis and mosquito’s death.
Another relevant idea is to produce ConA with a transmembrane domain. The aim is to use ConA as a receptor anchored in the membrane. This will go under more precise research.

Different plasmid for Asaia transformation can be used:
pNB92: secretion (neomycin resistance)
pHM2: replicative plasmids (kanamycin resistance), blue colonies phenotype when 5-bromo-4chloro-3indolyl-galactopyranoside is used.
pHM3: replicative plasmids (kanamycin resistant), blue phenotype.

The Human Practices group has worked to contact different industries and professionals to help us to move on our project and design.

Week 15 - May 31st to June 6th

The complete design of the project is getting clearer!

ConA will be produced in a dimeric conformation, a peptide signal will be fused to ConA and is detected by a secretion system, in order to be export through a protein system. The X protein will be fused on the other side on the other terminal side of ConA. It will either correspond to a protein able to change its conformation after the virus recognition, or a transmembrane domain.
The signalisation cascade is still under research. The latter will induce the activation of inhibition of negative or positive regulators.
The latter will regulate tTAV protein production and the Y protein (undetermined yet). tTAV needs to be produced in sufficient amounts to be lethal for the mosquito, and the Y protein will be produced with tTAV. When sufficient amounts of the Y protein will be produced it will induce the activation of a promoter controlling the production of a bacterial lysis protein. tTAV toxin will be then released within the mosquito’s gut and induce its death.

Week 16 - June 7th to 13th

The lab has now begun, six students are at the LISM in Joseph Aiguier’s CNRS: Rebecca, Liam, Yassmine, Fabian, Esteban, and Manon. Two researchers have been contacted by the Human Practices team to have an insight on biocontainment and biosecurity. A meeting will be organised in the next few weeks.

The scientific theoretical research is still going on:
Viral detection: In the idea to fuse a transmembrane domain to the ConA protein, an auto-transporter will be used. However, a huge amount of work on molecular modelling needs to be done to understand ConA conformation and binding domain, which could be involved in the interaction with the viral particle.
Signalisation: TonB system could be used. It is present on the external membrane in gram negative bacteria, and it allows the transition of the information into the cell.
Bacterial lysis and mosquito’s death: The aim is to do an inverse engineering for the construction of the operon responsible for the bacterial lysis and mosquito’s death. Indeed, in E. coli, a toxic protein (colicin) is produced and accumulates in the inner part of the bacteria. The latter induce the production of Holin (lysis protein) which has the role to lyse the bacteria and consequently release ColicinA, the toxic molecule, in the environment. The logic behind that is to replace ColicinA by CRY toxins, which are a good target to induce bacterial lysis. Indeed, it is present in Bacillus thurengiensis, and known to work efficiently. Indeed, when CRY concentration will reach the threshold, bacterial lysis will be induced, CRY will be released within the mosquito’s gut to induce its death.

Weeks 1 and 2 - June 3rd to 13th

As we wanted to create a genetically modified bacterium, we firstly had to think about our construction design. To do so, we did a lot of bibliography research, in order to find genes and plasmids that were going to be helpful. We have followed 4 different pathways:

The first one concerning the toxins we needed to kill infected mosquitoes,
The second one about ConA, as a detection system,
The third one deals with the transporter we found to help ConA going through the membrane,
The last one concerns the pRL1 plasmid, used as a timer system.

TOXINS - Cry-Cyt-p20
The toxins that we have decided to use in our project are : Cry11Aa and Cyt1Aa. These toxins are very interessant as they are working in synergy when it comes to toxicity. In addition, they have already been studied and show synergy.In fact, these toxins have also been tested on mosquitoes in the past, and it has been proved that they actually have deadly toxicity.
The bacterium needs to be protected from these toxins, that’s why we are also adding a chaperon protein : P20. This chaperon helps the bacterium stay alive and untouched by the toxic proteins (Cry11Aa and Cyt1Aa) by stabilizing it.
At this point, we were able to design the multiple sequences we wanted to try in order to succeed this part of the project.

ConA
After deciding we wanted to use ConA, we had to order its nucleotide sequence. Even if we learned that ConA works better when organized in tetramer, we made the choice to produce it as a dimer because in this form, the protein remains active and it is much more simple to produce. To optimize the interaction between the two monomers, we chose to add a sequence that codes for linker placed between the sequences coding for the monomers. We used a GSAT-linker present in the iGEM registry (BBa_K404301).

Week 17 - June 14th to 21st

During this meeting, we decided to not use tTAV, no protein X (unknown) will be fused to ConA, a two component system will not be ideal.
ConA export: The protein will be anchored in the membrane. For that, SEC machinery will be used, a signal peptide will be present on the N-terminal side of ConA, and cleavage site for peptidase will be found on the C-term. When ConA has passed through SEC machinery, it will be present in the periplasm and peptidases will cut the signal peptide. ConA and a hydrophobic domain (to determine) will remain. To pass the external membrane and have ConA anchored in it, we will add a coding sequence for Beta-Barrels. Secretion System T5a (SST5a) will be used to export and retain the protein of interest.
Viral detection and signalisation: As we cannot change ConA 3D conformation, the idea is to design a peptide with a N-terminal site encoding for a sequence able to interact with ConA, and, on the C-terminal side, a domain interacting with low affinity to ConA viral interaction site. The idea behind that, is to suppress the interaction between the design peptide and ConA when the protein sensor interacts with the viral protein. Consequently, C-term and the in-between sequence will be free for new interaction. The inner coding sequence is encoding for an interaction domain with a two-component system (TonB). As a result, the design peptide will interact with TonB, present on the external membrane of the bacteria, and induce a signalisation cascade.

Week 18 - June 18th to 27th

During this week, the business team has worked intensely to raise funds for the project.

To help the wetlab, the other part of the team is still under ongoing research.

For the signalisation pathway, TonB is still interesting for our project. Indeed, it is present within the external and internal membrane to capture and let pass proteins, mainly siderophores and iron. However, it is not known to recognize peptides. Moreover, there is still the specificity problem, as iron or siderophores particles can be present in the mosquito’s gut and can then interact with the TonB system.

For the lysis of the bacteria and the induction of the mosquito’s death, two main toxins have been chosen: Colicin A lysis protein (cal) and CRY. In E. coli, the colicin A toxin is present in an operon, and encoded by the gene from the same name. Within this operon, we can also find an immune protein and a lysing protein. The latter is responsible for a quasi-lysis of the bacteria inducing the release of the colicin toxin and the immune protein. This operon is regulated by a promoter containing an SOS BOX (induced when a DNA damage is detected). Our idea behind that is to construct a plasmid containing the lysing protein (cal), to induce bacterial lysis and CRY protein, which will be produced in higher quantities.

In order to control our system for the implementation, we have to find an efficient contention system. During the brainstorming, contention systems have been presented, such as: kill switch, optogenetic, CcdB and CcdA system (or toxin/anti-toxin), Geneguard system, Pt/HPt method, xenobiology, bacteriophage T7 RNA polymerase lysozyme system and R1 expression vector. Consequently, we can play with different parameters for the contention, but we need a significant parameter to be sure that the system will be used safely.

Week 3 and 4 - June 14th to 25th

Week 3

iGEM J04500

Transformations and MiniPrep GE Healthcare were made for iGEM J04500.

After transformation of iGEM plasmids J04500 we obtained two clones for J04500 (RBS + LacI)
A MiniPrep GE Healthcare was done on 3mL with the GE Healthcare kit. We measured with the Nanodrop (concentration, 280/260, 260/230) : (2) J04500 clone 1 : 37,44; 1,90; 1,60 and (3)J04500 clone 2 : 66,40; 1,87; 1,90.

iGEM I746916
After transformation of iGEM plasmid I746916, we obtained : 1 clone for I746916 (sfGFP). The MiniPrep GE Healthcare was done on 3mL with the GE Healthcare kit. We measured with the nanodrop (concentration, 280/260, 260/230) : (1) I746916 : 38,03; 1,91; 1,77.

PCR verification for iGEM I746916 and iGEM J04500:
For the PCR verification we used the oligos VF2 et VR (provided protocol). Then we did an agarose gel migration. The awaited band 600 ~700 pB for J04500 was good, but the band for I746916 is twice larger than the awaited one (2000pB vs 1000 awaited). Thus, 2 hypotheses : either there was a problem and it is not the right strain, or the plasmid DNA has not been linearized and gonna migrate less further than expected, the length is not gonna be the one expected.

E1010, E0040:
We have used plasmids from iGEM 2020. Parts E1010, E0040 with : RFP: Red Fluorescent Protein et GFP (mut3b).

Colony PCR, E1010, E0040:
We did a colony PCR and a verification of E1010 and E0040 following the protocol EconoTaq PLUS GREEN 2X mm, bands have migrated well at the awaited sizes.

Week 4

Transformation sfGFP (part I746916) 2018:
The transformation did not work. The project of cloning with sfGFP has been abandoned. We will use GFP which is less efficient to approach the periplasm but functional.

PCR on colonie, part E1010, part E0040, pRL1:
Purified plasmids (E1010 and E0040) will still be used to amplify GFP and RFP and thus use it later for the insertion into the plasmid.

Amplification PCR: 1. GFP : part iGEM control with iGEM primer
2. GFP : primer colicine
3. RFP : part iGEM control with iGEM primer
4. RFP-Col

The expected bads were found:
1. GFP iGEM: 700bp
2. GFP Col: 700bp
3. RFP iGEM: 700bp
4. RFP Col: 700bp

Restriction Ligation:

Digestion pRL1 with BlpI and BssHII
Digestion GFP-col with BlpI and BssHII
Digestion of plasmid lacI-RBS with Spe1 and Pst1HF
Digestion of GFP with Xba1 and Pst1
Digestion pRL1 with BssHII and Blp1

An amplification of Pref-6His-ConA-linker-BamHI was also done.
We did a colony PCR to verify the results of cloning for RBS-lacI and the bacteria have integrated RBS-lacI and GFP.

Test of the product PCR of Pref-6His-ConA-linker-BamHI.

We obtained a band at about 1000 bp for an expected size of 1020 bp. We can conclude that the PCR worked. There has been amplification of Pref-6His-ConA-linker-BamHI.

Test of the product PCR of Pref-6His-ConA-linker-BamHI-Suffix

We obtained a band at about 1000 bp for an expected size of 1050 bp. We can conclude that the PCR worked. There has been amplification of Pref-6His-ConA-linker-BamHI-Suffix.

Week 19 - June 28th to July 4th

We have found a wiki programmer: Johan Hascoet. He will help us to build the wiki for this year!
We have studied different wiki from a worldwide team, to have more ideas to create our website for the ARBO-BLOCK project. During this meeting we have mainly worked on the design, the choice of the colour to use in our wiki, linked with the ones that we’ve already used for our social network pages.

Week 20 - July 5th to 11th

It has been a month that the wet lab team is working at the LISM in the CNRS. They are busy with the practical work and are now the main group working on research.
On the other side of the lab:
Stalker team is paying attention to the updated rules for the deliverables/meeting/medal requirements.
Businesswoman and Businessmen have now filled up and sent all the form and financial files. They are now preparing a presentation to introduce the iGEM competition and 2021’s project to the partnership.
Team Events is now in charge of organizing with Parisian’s teams a French meet-up. The latter seems to be organized in presential, in Paris. Alizé, Manon and Aldo will be actively involved in this meeting organization.
Under the active Human Practice Team, we wanted to organize a street interview to collect the vision and point of view of the population from Marseille on our project. For this purpose, we need to make and print flyers, create a google form in French and English to have interpretable data.

The promotion video is now ready!

Week 5 and 6 - June 28th to July 2nd

Week 5

Transformation and colony PCR for PCR verification, following the protocol EconoTaq
PLUS GREEN 2X mm : Flag-Cry11Aa pBAD24: migrated at the expected size ; Strep-Cyt1Aa pBAD24 : migrated at the expected size ; His-P20 Flag-Cry11Aa pBAD24 : migrated at the expected size. We did a digestion of plasmid lacI-RBS with Pst1HF and Spe1 and a digestion of Pref-6His-ConA-linker-BamHI-Suffix with Xba1 and Pst1. The transformation of the ligation Prl1-GFP with NEB5-alpha competent (bioLabs) and the transformation of the ligation Prl1-GFP with 5-alpha competent have worked, a lot of colonies are visible, a Colony PCR was done to verify the results of cloning.

The three plasmids will be sequenced.
All the constructions with pRL1-GFP were wrong due to the absence of RBS. We restart all the manipulations involving pRL1-GFP.

Sequencing:
After the digestion analysis by PCR we chose 3 clones from 3 different cloning. These 3 clones will be sequencing to be sure to don’t have any mutation in the toxin sequence.
The results of the SLIC Cloning pBAD24 P20 were successful. In fact, a clone was obtained from the cloning vector (pBAD24) + insert (P20). Following the Econotaq PCR colony protocol, the expected size was 630pB, a digestion profile will be done.

Week 6

A Mini-prep on the starter of RBS-lacI-GFP was done following the GE healthcare protocol and a digestion of the RBS-lacI-GFP plasmid with XbaI and PstI. We obtained some sequencing results : For pBAD24_cyt1Aa, sequencing pBAD24fw no mutation is visible, the entire gene was sequenced. These clones will be transformed into E. coli MG1655. For the results of the SLIC Cloning for pBAD24_P20, a digestion profil was done, and a clone was chosen for sequencing. A Digestion of plasmid lacI-RBS with Spe1 and Pst1HF, and a digestion of Pref-6His-ConA-linker-BamHI-Suffix with Xba1 and Pst1HF were done.
For the colony PCR on PelB-sfGFP the results were positive, in fact, the clones had integrated the PelB-sfGFP insert. We sent pBAD24_P20_Cry11Aa for sequencing. A Digestion of the PCR results (pRL1-RFP) were done with Dpn1 to suppress the native plasmid. The transformation of pBAD24_cry11Aa in E. coli MG1655 was successful, which means that these colonies can be used to express our toxin. For pBAD24-p20-Cry11Aa in DH5a, a mini-prep on the starters and a restriction profil with EcoRi and Pst1 were done. Then the results were sequenced.

For pBAD24-p20-Cry11Aa in DH5a
At this step we have some information about the expression. After 3h of incubation, the OD600 = 8.73. This OD is 2 times more important than toxin expression where OD600 = +/- 4.5 for E. coli MG1655 cry11Aa and E. coli MG1655 cyt1Aa. That means that the toxin expression is toxic for the bacteria, or it makes the growth slow. For the chaperon expression (P20), the OD600 = 5.5, the expression decreases bacterial growth, but we can’t really compare because we are in E. coli DH5a. For the result of sequencing pBAD24_P20 no mutation was visible, the whole gene was sequenced. The sequence is good. A PCR were done to set the suffix to Pref-6His-ConA-linker-BamHI, the digestion check was good: The expected size was at 1050 bp and our results were about 1000 bp. We conclude that there has been amplification of Pref-6His-ConA-linker-BamHI-Suffix.
Three digestions were done: Digestion of plasmid lacI-RBS with Spe1 and pST1HF, digestion of Pref-6His-ConA-linker-BamHI-Suffix with Xba1 and Pst1HF and Digestion of J04500+GFP with SpeI/XbaI in 3 conditions : XbaI/SpeI ; SpeI and XbaI.
Miniprep was also done on starters containing pBAD24 + P20_Cry11Aa and J04500+GFP.

Week 21 - July 12th to 18th

The Events pole is working hard to find festivals to attend close to Marseille.
We have been accepted to have a stand and present our project at the “Festival des Sciences” which will take place in Marseille in October. Additionally, we will have a slot at the “Forum des Biotechnology” organized by the engineering school Polytech Marseille, to present our ARBO-BLOCK project to engineer students.
With all those events coming closer, we need to have our logo. Members of the team have proposed different designs and we also called Studio Emera, held by Justine to propose a design. It will be sent to the team and a decision will be made during next week's meeting. Finally, the team is working on the preliminary safety form, a deliverable needed to stay in the competition.

Week 22 - July 19th to 25th

Everything is going well at the lab, the team is working hard even if they encounter problems with the delivery.

The Business team received validation for subvention from different companies. They are now working on all the administrative work.
The Human Practices team has difficulties reaching associations and organizations to arrange a meeting, as they do not answer by phone or email. They will keep going!
We finally have our logo for the ARBO-BLOCK projects by Team Aix Marseille University!

Week 7 and 8 - July 12th to 16th

Week 7

Toxins expression
Western blot : E. coli MG1655 pBAD24_cry11Aa, E. coli MG1655 pBAD24_cyt1Aa, E. coli MG1655 pBAD24 (control), E. coli DH5a pBAD24_P20.

The Transformation of pBAD24_P20 in E. coli MG1655 was successful. We had to test the product PCR of Pref-6His-ConA-linker-BamHI-Suffix and we have noticed that the amplification of Pref-6His-ConA-linker-BamHI-Suffix has been done. Another transformation of J04500 PelB sfGFP and J04500 LacI RBS was made. We revealed the western Blot : 3 conditions were studied 6-His tag, Flag and Strep tag, repeating the protocol for each condition. We obtained flags (Cry11Aa), 6-his (P-20) and strep (Cyt1Aa). A SLIC and a transformation of pBAD24 + PelB_sfGFP_Tranportor was made. Colony PCR that was done on pLacI/ RBS + ConA showed an expected band for plasmids ConA-Linker (1540 pb). We obtained a clone that corresponds to pLacI/ rbs = ConA linker. The Sequencing result for this week was done : Fw pBAD24_P20_cry11Aa (Using primer : pBAD-FP) has no mutation. The digestion of pBAD24 with NcoI was successful, a band at 4500pB is visible : pBAD24 was restricted by NcoI. We obtained clones for pRL1-GFP by PCR screening.

Week 8

We made a colony-PCR to verify if the colonies we obtained after transformation of GFP-ColA construction contained a sequence with a length corresponding to what we expect. We obtained 3 clones with bands at the expected size : clones 5, 11 and 18.
We made liquid cultures of bacteria containing plasmids with GFP-ColA or RFP-lys constructions in pRL1 before purifying them with miniprep.
We then digested the purified plasmids with SacI/BssHII (for pRL1_GFP-ColA constructs) or NcoI/BamHI (for pRL1_RFP-lys).
After migration on an agarose gel, we expect to observe a band at 730pb for the plasmids containing GFP-ColA and a band at nearby 1800pb for the plasmids containing RFP-lys.
We obtained one condition with a band at the expected length for GFP-ColA constructs (clone 18) and two conditions with a band at the expected length for RFP-lys (clones 11 and 12).
As the verifications we made seemed to give good results about the insertion of our sequences at the positions wanted in pRL1, we sent to sequencing the clone 18 for the construction GFP-ColA and the clone 11 for the condition RFP-Lys.
We prepared the stock solution of Mitomycin C at 300µg/µL that will be used for further experiments.
We made a 5mL starter of the GFP-ColA clone 18 / RFP-Lys clone 11 in DH5a E. coli strains to measure the fluorescence level with and without MitomycinC induction but we forgot to add antibiotic after adding our bacterias that had grown in a new liquid medium.
We realized the calibration curves with fluorescein and TexasRed, that will allow us to normalize our fluorescence-experiments results ; by measuring the fluorescence level recorded by the TECAN machine with different gains and with different quantities of fluorescent molecule in the well.
We made a colony PCR on the two clones we obtained for pBAD24_PelBsfGFP_AidaI with two oligonucleotides couples : ebn158/o2129 omp82/83. With each oligonucleotides couple, we obtained a band at expected length after migration of the PCR products on an agarose gel.
We did the IPTG induction test on our DH5a E. coli bacteria containing J04500_PelBsfGFP and empty J04500. We determined that the Lac promoter contained in the part BBa_J04500 was not responding to IPTG induction in our bacteria.
We ordered primer sequences used to realize our pBAD24_p20_cry11Aa_cyt1Aa construction by SLIC.
We made liquid cultures of bacteria containing different conditions (pBAD24 empty ; pBAD24_6HisT7 ; pBAD24_P20 ; pBAD24_cry11Aa ; pBAD24_cyt1Aa ; pBAD24_P20_cry11Aa) to
We followed overproduction protocol on the bacterias cultured in a liquid medium and added bME and Buffer to prepare samples for SDS-PAGE.
We prepared an SDS-PAGE gel, charged it with the samples we prepared before and then ran the electrophoresis. We then followed the Western Blot protocol using Anti-His antibodies ; or Anti-Flag + AntiStrep antibodies to reveal our P20 ; Cyt1Aa and Cry11Aa.

Week 23 - July 26th to August 1st

The Events Team managed to get us a stand at the Lumexplore Festival!
It will take place in La Ciotat from the 16th to the 19th of September.
The organization for the French Meet-up is still in progress.
Other fundings has been accepted and sent to the treasure.
We have found a costume for our mascot: THE MOSQUITO!

Week 24 to 27 - August 2nd to 29th

The French Meet-up will be held online during the 11th of September. The French team will have to present their project, a workshop will be organized, and stakeholders will be invited.

An Instagram game will be launched soon: Teams from this 2021-year competition will send us a description of their project thanks to EMOJI! The description will be put in our Instagram story and our followers will have to guess at which team this description belongs to. The winner will get a goodies bag!
As we now have events to participate in, we need to take care of the goodies for the team and for the public. The team is choosing which goodies they want.
The French Meet-up is coming closer, and we need to prepare a presentation to recap our work during those 6 months, in 10 minutes time. The experimental work is now finished. They will present their results to the whole team soon!

Week 9 to 10 - July 26th to August 6th

Week 9

For the toxin expression the results were not sufficiently conclusive, so a western blot was done with the preserved samples of the constructs: E. coli MG1655 pBAD24_6hisP20, E. coli MG1655 pBAD24_flagcry11Aa, E. coli MG1655 pBAD24_strepcyt1Aa
Potentially P20 had transferred badly during the blot. The antibodies were also well separated to be sure not to have specific labeling (because bands overlapped).

A Mini Prep of pRL-LysRFP and pRL-GFPcolA were done and the restrictions of:
Restriction of sfGFP by XbaI and PstI-HF, Restriction of J04500 by SpeI and PstI-HF and Restriction of ConA by XbaI and PstI-HF.

A transformation of pBAD24_PelB_sfGFP_AidaI in MG1655 and a transformation of J04500 + SfGFP and ConAwere done.

We studied the expression of toxin : E. coli MG1655 pBAD24_6hisP20, E. coli MG1655 pBAD24_flagcry11Aa and E. coli MG1655 pBAD24_strepcyt1Aa
Finally, for the production of Cry11Aa and Cyt1Aa, we can clearly see the expected bands (~ 72kDa, ~ 28kDa respectively). For the production of P20,no bands were observed.

The amplification PCR of GFP with the new primers were studied and we found the expected size 750 pb. A digestion of pRL1 - RFP with SacI and BssHII was done.

A Growth kinetics and the expression as a function of time: The equivalent of 1 UDO was sampled at each OD measurement from induction to be able to analyze protein expression.

E. coli MG1655 pBAD24_6hisP20: For the construct allowing expression of P20, again P20 is not visible with anti-histidine antibodies. However, P20 is clearly visible in the construction allowing the expression of P20 + Cry11Aa. That could mean that P20 in this construct is in fact mutated on its coding gene and would make it a 1kDa protein. To ensure that it is well mutated, the reverse sequencing will be done with oligo ebm159 on clone II. If the result is not convincing, clone I will be sequenced.
E. coli MG1655 pBAD24_flagcry11Aa: For the Cry11Aa expression construct, finally there seems to be bands showing expression over time, but these bands are not of the expected size (ie 72kDa). This is one we are still waiting for the result of reverse sequencing. Nevertheless, these results (potentially truncated form of Cry11Aa) are reproducible, this is not the first time we have seen a band of this size.
E. coli MG1655 pBAD24_6hisP20_flagcry11Aa: For the construction allowing expression of P20 and Cry11Aa. On one hand P20 is present but we find it a little higher than expected (maybe normal). On the other hand it is not expressed over time, after induction it directly seems to be at its maximum concentration. Cry11Aa seems to be present but we do not really see the expression over time.
E. coli MG1655 pBAD24_strepcyt1Aa: For the construction allowing expression of Cyt1Aa, we clearly see over time an expression which seems to be increasing. This is the only construct that did not have any ambiguity in its sequencing. It migrates well at the expected size (28kDa)/

A Digestion of pRL1 - RFP with SacI and BssHII was done.

Ligation GFP + Lys-RFP

We did the preparation of mitomycin C.

Ligation of the restricted DNA sfGFP and conA : We used : Restricted ConA, Restricted sfGFP and Restricted J04500 Ligation of sfGFP in J04500 and conA in J04500 : Transformation of sfGFP in J04500 and conA in J04500 on dh5alpha. And we have done a transformation on the previous sfGFP in J04500 and conA on dh5alpha.

Sequencing result: pBAD24_6hisp20_flagcry11Aa : The reverse of the genes encoding Cry11Aa in pBAD24_P20_cry11Aa has a mutation at the end. Another clone will be sequenced. pBAD24_flagcry11Aa : The reverse of the gene encoding Cry11Aa in pBAD24_cry11Aa cannot be analyzed. pBAD24_6hisp20 : The fw of gene encoding P20 in the pBAD24_P20 construct had an ambiguous mutation.

Restriction of the amplified DNA sfGFP + conA on J04500 plasmid ; Restriction of sfGFP by XbaI and PstI-HF ; Restriction of J04500 by SpeI and PstI-HF ; Restriction of ConA by XbaI and PstI-HF.

Week 10

A BSA range was done in order to quantify the protein in agarose gels.
From culture of MG1655-pRL1,MG1655-ColA-GFP and MG1655-RFP-lyse : OD (600 nm) and fluorescence measurement to Tecan every 30 minutes during 5 hours, after an overnight culture and mythomicin induction.
Miniprep were done on : DH5a pGFP + RFP 4, DH5a pGFP + RFP 5, DH5a pGFP + RFP 6, DH5a pRFP + GFP C, DH5a pRFP + GFP D, DH5a pRFP + GFP B, DH5a pBAD24 cry11Aa clone 3, DH5a J04500 clone 2 and DH5a pGFP
In addition, agarose gels were done and the results were satisfying for the clones C, D and B but not for the DH5a pGFP. We did RFP and GFP measurement another time in order to have experiments done twice at least.
Culture of pBAD24-6his-P20 clone 2 ; pBAD24-6his-P20-flagCry11Aa clone and pBAD24-flagCry11Aa clone 2 in 2YT medium and ampicillin, to select the resistant ones. These cultures will allow us to do Western Blots in order to verify their expression.
Then we did an over production test for Cyt1Aa : changing the temperature of culture, the time of culture, and the percentage of arabinose induction. We actually did this twice to have exploitable values. Then, we did a western blot to see where there is expression depending on the different conditions.
We also did ligations of sfGFP in JO4500, and conA in J04500.
Amplification PCR (for the SLIC) of conA : we had a band at 900bp, it is the awaited profil of conA. Then, we were able to do the SLIC : pBAD24-conA.
After that, we did a colony PCR on pBAD24-conA and obtained the awaited band for every colony.
We also followed the OD and the fluorescence of MG1655_pBAD24_PelBsfGFP_AidaI in different conditions : depending on arabinose concentration and the time of induction.

Week 28 - August 30th to September 5th

We are now focusing on the wiki, the different sections, and under-sections in it, what we really have to focus on, and divide the work between the different members of the team.

The goodies have been ordered to Wiloo company and received!

The team participated in the welcoming day for the new student from Marseille Science University during the 2nd and the 3rd of September. The presentation for the French Meet-up is almost done, we will be ready to present our project to the participating teams.

Week 29 - September 6th to 12th

The French Meet-up got on very well on Saturday! The team was overjoyed to participate and have the chance to meet other teams. The workshop was very constructive and helpful for our project and its management.

We have now to focus on the next event coming: LUMEXPLORE FESTIVAL. We will have a stand for the weekend. As it is a festival destined to the entire population, besides presenting our project and collecting people’s opinion on it, we wanted to sensitize youths to security and equipment in the lab. For that, we will set up an experiment to extract and visualize DNA from fruits, and “The scientific mystery box.” Within the latter, we will place material commonly used in the lab, by touching and guessing the 3D structure, they will have to guess what is inside the box!

Finally, and most important part, different groups are working on the deliverables: safety form, preliminary judging form, abstract and title.

Week 11 and 12 - August 9th to 20th

Week 11

Another OD and fluorescence measurement of MG1655-pRFP, MG1655-pGFP, MG1655-pRL1 and MG1655-pGFP-RFP at DO = 0,4, then we sent them to sequencing.
Digestion profil of clones 1, 2 and 3 of conA expected as good ones : they are all good as they show the band at 900bp. Then transformation of conA in MG1655 and protein expression test on conA. Expression test for repression of J04500.
SLIC of Cry11Aa in pBAD24-P20-Cyt1Aa. This is the final sequence of our toxins sequence goal.
Order of the sequence of Cyt1Aa already registered in iGEM parts and PCR to do the SLIC in pBAD24. Then we did a transformation in DH5a
Megapriming of pRL1 and then transformation in DH5a
We made many attempts of IPTG or Glucose incubation in minimal mediums M9 to determine whether the lack of protein production in presence of IPTG and the diminution of protein production in presence of glucose are not due to elements that could be present in the LB medium but we never obtained good results.

Week 12

SDS-PAGE analysis of the MG1655-pRFP, MG1655-pGFP, MG1655-pRL1 and MG1655-pGFP-RFP at DO = 0,4, experiment.
Purification of Cyt1Aa as the sequencing results were good. The different fractions retained were analysed by SDS-PAGE and Western blot.
To continue with the toxins we did some SLICs in pBAD24 either with 6his-P20-flag-CRY11Aa-strep-Cyt1Aa (final construction goal) or with Cyt1Aa (from iGEM part bank), finally, we transformed them in E. coli. Then we did colony PCR after overnight culture in order to verify that our transformation worked.
We did another expression test but this time with conA in pBAD24 transformed into E. coli MG1655. Then we did a SDS-PAGE and a Western blot to reveal the expression of this construction.
Amplification PCR on the new transporter - aidA1 - sequence. SLIC transformation into E. coli DH5a bacteria.
Another OD and fluorescence measurement of MG1655-pRFP, MG1655-pGFP, MG1655-pRL1 and MG1655-pGFP-RFP at DO = 0,4 - measurement with the TECAN and analyze with SDS-PAGE.

Week 30 - September 13th to 19th

The Lumexplore Festival was a real success! The team was able to present the project and collect different visions from the population on it. Children and teenagers were really interested in the activity we proposed! Our mascot is now famous and caught everyone’s attention. We are now checking the judging form, abstract, parts and safety form all together, to have everyone’s opinion and corrections.

Finally, we are setting up the presentation video. We are brainstorming to collect ideas to realize the script and the video.

Week 31 - September 20th to 26th

The safety form and the various parts are now ready and registered on the website. The whole team is working intensively on the wiki.

Week 13 and 14 - August 23th to 31st

Week 13

We made a directed mutagenesis to suppress an XbaI site present in a sequence that we wanted to present as a part in the iGEM registry. We made an SDS-PAGE with samples having different constructions, and induced with mitomycin C or not.

We started overproduction as if we were going to test them on blood cells and living mosquitoes. To obtain a sufficient quantity of toxin, we started a liquid culture of bacteria containing pBAD_cyt1Aa in a volume of 2L of LB. After that, we purified our toxin, and kept an aliquot of every fraction of elution (E0 to E3) for analysis by Western Blot.

We finally obtained a construction containing P20 and the Cry11Aa and Cyt1Aa toxins in pBAD24.

We tried again to clone the sequence for aida-I coming from the iGEM registry in order to make the same experiments as we did with our aida-I sequence to fill a Gold Medal Criteria. We amplified it by PCR using o2148 and o2149 primers and followed the SLIC protocol to insert it in pBAD24 with pelBsfGFP. Unfortunately, we still did not obtain clones after transformation of the plasmid in competent DH5a E. coli bacteria.

We also tried one more time to clone aidaI (with mutation) with pelBsfGFP in pBAD24 with SLIC. We obtained two colonies but due to a lack of time we decided to put the bacteria in liquid culture to perform a Western Blot to conclude about the presence of sfGFP.

Week 14

We tried one more time to determine the differential fluorescence intensity evolution in our construction gfp-colA+rfp-Lys with induction by mitomycin C, measurements at different times and preparing samples for SDS-PAGE.

We also made the last SDS-PAGE and western blot for J04500_pelBsfGFP, pBAD24_pelBsfGFP_aidaI and pBAD24_pelBsfGFP_aidaI (with mutation) to localize whether the GFP is localized in the cell or in the extracellular medium. We centrifuged our cells and loaded both the bacterial pellet and the supernatant onto SDS-PAGE gel. We also realized SDS-PAGE and western blot with antibodies directed against P20 ; Cyt1Aa and Cry11 Aa.

Week 32 - September 27th to October 3rd

Another event has been organized on the 30th of September at Aix Marseille University. We have been able to present our project, collect more data, and also to begin the recruitment for next year's team! It was very interesting for us to face scientific students mainly in Master's studies. They were very interested by the project but also by the iGEM competition!

Week 33 - October 4th to 10th

During this week-end, members of the team participated in “La Fête Des Sciences” in Marseille. It is a really famous science festival. As at the lumexplore festival, we were able to collect data, share our knowledge and love for science. We have met around 300 people, and a lot of children. They were all very interested in our activities : the mystery box and DNA extraction. Fabian, Yassmine and Nicola were really happy about this event as they have met many children and their parents, but also scientists!

Week 34 - October 11th to 17th

The end of the project is getting closer. The wiki and the presentation video are getting ready!

We were able to have a new intervention with a researcher specialized in virology: Dr. Bruno Coutard. We exchange a lot on arbovirus diseases and emergence. He was really enthusiastic about our project! Thanks to this exchange we were able to rethink our ARBO-BLOCK design. Dr. Coutard suggest us to work with vHH (small antibody fragment) in order to provide an arboviral-specific system! This will allow us to secure our construct and to be even more specific with high affinity. It will be taken into account for the next step part of the project.

Week 35 - October 18th to 21st

This is the last week before WikiFreeze! Everything is getting ready, and the presentation video also!

Team:Aix-Marseille/notebook