Team:HebrewU/notebookdates.html

March 1st.

March 2nd.

March 3rd.

March 4th.

March 5th.

March 6th.

• Group Meeting in Jerusalem (Ein Karem + Givat Ram)
  Recruit students for our project from the faculties of computer enineering and medicine.
• Lior B, Lea, Noa and Alon.

March 7th.

• Meeting with Tomer Malchi (CultivAid, The Hebrewu University)
  Tomer mentioned the KOW value (hydro/lipo-philic qualities, polarity) which can be used to predict how well molecules will be able to diffuse through root membranes.
  Molecules that are between 2-3 can penetrate easily through the membrane while higher or lower values may have trouble:
  
  
  a. Molecules that are bigger than 500 D will also have trouble passing through the membrane.
  b. Molecules that have net charge can't [easily/passively] pass passively through the membrane.
  c. Molecules PKA effects the charge, and as such soil pH can play a role in determining uptake(causing the plant to affect pH may help us uptake molecules).
  
  
  Specifically, TCDD has KOW of 4.5 meaning it won't enter a plant in a passively. When we checked the dechlorinated version of the molecule we saw that its KOW is 3 meaning the plant should be able to absorb it [passively].
  
  Moreover, Tomer suggested to check if we can enhance oxygenation in soil and by doing so, we can boost the degradation of different compounds.
  
  When Tomer was asked about different methods to measure how well a plant absorbed/ broke down a certain molecule he said that we the machinery to check how well a chemical is present in soil solution or plant solution he said that we need to check if someone made a protocol for it before. If we can find a protocol, we probably have the machinery needed in the faculty (HPLC chromatography)
  Further on Tomer mentioned that he has a special pump that can measure the ratio between the concentration of chemical in plant phloem to the solution that the plant was based in (while using aquaponics).
  
  Furthermore, we understood that in order to build a system to detoxify TCDD in Vietnamese soil we need to pay attention to the soil properties (clay % and organic matter concentration).
  Tomer recommended to discuss our idea with Baruch Robin, professor from the agriculture faculty that work in the field of herbicides.
  Also, Tomer recommended to discuss our idea with Itzhack Hadar, professor from the agriculture faculty that deals with white rot fungi that can break down different chemicals (Without the need to pass them through a biologic membrane).
  
  When Tomer was asked about the possibility of cleaning the water from the toxin, he recommended to check "Constructed wetlands".
  
• Alon, Amichai, Aliza, Omri, Shai, Eliya

March 8th.

• Whole Team Meeting, Discussing the near Future with Assaf and Shai (Mentors).
  Meeting summary:
  
  - We couldn’t find the sequnce of TCDD degradation enzymes that we found in the literture.
  - Shai (Metnor) said that we should contact labs that already showed lever cells that degrade any kind of dioxin and see if they can give us the specific enzymes that were part of the degradation.
  - Do the same thing as above with the recombinant E.coli (from one of lior`s articles) (when we ask this kind of things we should ask for everything, the Ecoli and all the plasmids with the enzymes and the DNA sequences and start checking how much time and money it would take to express them ourselves) we should ask if they tried TCDD as well.
  - We should check references of the article with 3 enzymes in other articles and see what others had to say about it.
  - We should summaries all the candidates for degradation, what kind of reactions we know they do and think how we can check the products of TCDD degradation by them. What the intake and out take of the prosses as well (not just the substrate), maybe we can use it to create a test for degradation.
  - Maybe looking for agent orange, and check where in the degradation prosses it becomes detoxify and the enzymes relevant for that.
  - Send a mail to the beijing team to understand their work.
  - Mosquna and shai says that trying to understand the uptake of TCDD to the plant is a big task.
  - If we start researching the uptake by plants we should add it to our project even if we weren`t able to combine the degradation and uptake in the same plant.
  - Shai expresses his positivity in adding the arabidopsys to the project.
  - Mosquna says that when they want to check something that it works the inject agrobacterium to the leaves of tobacco.
  - We need to grow the arabidopsis in advance to shorten the time frame of every generation.
  - Fungi has a much better uptake then arabidopsis so it might be better.
  - We should send mails to all relevant labs.
  - Maybe we can search for a place they already have full grown symbiotic mushrooms (talk with tomer again).
  - When we read articles, we should care much about concentration.
  - If we use the fungi, maybe we can find a relevant assay for the connection between the fungi and plant.
  - Assaf (Mentor) said that we should concentrate on the next topics when we read articles:
  
  ○ Enzymes.
  ○ What kind of reactions.
  ○ What are the products.
  ○ How can we detect and follow the reaction prosses.
  
  - We might be able to do a research with bacteria to create bacteria that can live on TCDD and mutagenesis. Mosquna sent us an article about the method of finding the bacteria that prosses the T4D. Mosquna says he has a friend that deals with this kind of things so he might ask him for bacteria populations.
  - We should check if someone knows about a bacteria that knows to use dioxins as carbon sources.
  

• Everyone.

March 9th.

March 10th.

March 11th.

March 12th.

March 13th.

• Library Meeting with Assaf and Shai(Mentors)
  - We reviewed all the important dates for lab work.
  - We outlined the lab work.
  - Mosquna said that we need to check what the enzymes we found need to work (if transferring them from bacteria to plants will work).
  - All anaerobic enzymes may not work in aerobic organisms.
  - Red team should be divided in the different enzymes. (we might ask the help of our associated students).
  Red team should check:
  
  - Do the enzymes work in the context of our organisms?
  - Do they need any other enzymes to work?
  - Do they work in plants and bacteria as well?
  
  - We should look again for a way to break the products to CO2 and water.
  - We need to reconstruct the 4 experimental pathways to start constructing our experiments.
  - Regarding the assay, mosquna thinks we should check the formation of our product to test the enzymes.
  - We should check how much the Aliza and Hagit proposes cost and where we can get it.
  - We should look for NEB sponsorship.
  - We should check more thoroughly the use of biobricks and DNA in the competition.
  - We should decide what parts we want from igem before we receive the biobricks kit.
  - Addgene can send us vectors needed.
  - Igem parts might not be reliable enough.
  - Mosquna wants to be part of the choosing of vectors.
  - Computational biology - use them for a model of the plants work beforehand.
  - How and where exactly the enzymes work (coenzymes, cofactors, where in the cell they work, what kind of environment they need).
  - We should check what receptors we know for TCDD and its products to create dependency in plant and bacteria. (we can use it as a kill switch and an assay).
  - We should write a vision of all the genes we would like to add to complete the TCDD cycle.
  - We can't rely on the faculty time table, they will react too slow. Mosquna will give us a banch in his lab.
  - We should check if AHR exist in E.coli and Arabidopsis.
  - If AHR exist in yeast already, we should order it now.
  • We should contact the guy from givat ram and get his E.coli.
  - We should look again at all the old articles we have and look if anyone conducted more work later on.
  - Mosquna will contact berta and talk about how to conduct orders.
  - What regulation exist for working with TCDD.
  - We will conduct another meeting in Sunday after the conference in Weizmann.
  - We should search for an industrial multigene vector.
  
• Everyone.

March 14th.

March 15th.

March 16th.

March 17th.

March 18th.

• Synthetic Biology Conference in Weizmann
  The conference included lectures, round table discussion and a showcase of former I-GEM project from Technion, Tel Hai and Beer Sheva teams.
  
  
  
  
  
  
  
  
  
• Everyone.

• Team Meeting - Funds and Research
  We reviewed all the numbers that we got to see if we can guess our budget for research.
  - Assaf (Mentor) think we will need about 50,000$ for research alone, including 15,000$ DNA synthesis.
  - There is 20kb from IDT - https://2018.igem.org/Sponsors/Special_Offers/IDT.
  - Twist might be able to aid us in funding with DNA synthesis or cash.
  After reviewing our current information, we concluded that:
  
  1. PR has to do research to understand where TCDD is located and considered a problem to guess which companies and organizations will be contacted for support.
  2. Write a scheme of our proposed solution for marketing reasons (just the idea, not much details, focus on problem and end results). Magnify the problem (it`s around the world, show a lot of research that support that, how bad it is for human health and environment). Show a simple figure of how the solution works.
  3. PR team should contact Berta and arrange a meeting to regulate the donation searching prosses.
  4. We should find a reductase in yeast cell for the cyp metabolic pathway. Plus we should look at people who tried to express this kind of enzymes in general in yeasts and see what they did and what worked best.
  5. We should also look how the fungi extract the enzymes.
  
  
  Organizations that popped up during the meeting:
  
  1. Veterans of Vietnam.
  2. Kakal.
  3. Bill gates.
  4. Jews from the states.
  
  Lab space:
  
  - We have a small lab desk but we have no equipment, Assaf says we will work in his lab for now.
  - Plus he says his doctors will be our mentors and help us with the lab work.
  
• Everyone.

March 19th.

March 20th.

March 21st.

March 22nd.

• Team Meeting about the Enzymatic Pathways.
  We discussed the first enzymatic pathway that we found in the literture.
  
  - For now we concluded that we need a reductase for NADPH to recycle the prosses.
  - We can degrade the tetra to tri, there are a few enzymes that we can add to degrade the tri (including other mutations of cyp1a1.
  - There is a site called "addgene" that cells plasmids and viruses so we might find what we need there.
  - We might try to send the cyp crystallography to Weizmann to analyze it and improve it.
  - We should find a candidate with the most structural information and send it to them.
  - We should choose one of the cyps that we think that might work for us, find structural information and send it to Weizmann and try to improve its specificity to TCDD and maybe even degrade it further then triCDD.
  
  
  Asaf presented his idea for experiments:
  
  - Use a yeast vector with a promotor called "gal1-gal10" that can provide for two genes at the same time.
  - When we introduce it to the yeast, we can merge different yeasts with different promotors (they merge their genetic information) and so we can get more enzymes in 1 yeast.
  - If we could find an assay for the products that is quick and cheap, we might use a total random reactions and collect the best yeasts.
  
  
  We presented another product of a reaction that is pyrocatechol which is toxic for human. We need to check if we can work with it, it has good assays.We concluded that every assay for potential products or byproducts of the system is worth doing.
  
  We discussed the second enzymatic pathway:
  
  - We concluded that we might get from TCDD to catechol which every plant can degrade.
  - It seems that we can use TCDD as a carbon source, which is easy to check.
  - Any way we need a reductase for NADH/NADPH (depends on the enzyme).
  - Cyp1 might help this enzymatic pathway as a Cl ridder.
  
  We discussed the forth enzymatic pathway:
  - With a dioxin without the Cl`s atoms being degraded by two enzymes to catechol.
  - There are more enzymes that might be easier to use then the second enzymatic pathway. (we should combine the pathways).
  - We should send a mail to the Chinese research team, and maybe even try their sequence ourselves.
  
  We discussed the third enzymatic pathway.
  
  We concluded that all 4 enzymatic pathways are viable solution.
  
  - We need to optimize the sequences we found for yeasts.
  - Because we want to use plants in the future, we want to use codons that are good for plants and yeasts.
  - if it takes too much time (more than an hour) we should go for yeasts optimization alone.
  - We should be able to do that in sequencing company's sites.
  - Enzyme names are not Absolut! We should use the exertion numbers of each gene.
  - We should check if there are splices to each gene.
  - When we have those sequences we should check with IDT and twist to get price tag and supply timeline.
  - We need to contact twist and IDT and see if we can get the sequences.
  - We have a notebook of the yeast vectors to read and understand how to work with.
  
  
  We found out that there are 2 options to insert the genes to the yeasts:
  
  1. Plasmids, which will give us more enzymes in the cells but the difference between cells is high (10,000 times more enzymes between cells).
  2. We can insert it to the yeast`s genome which will be more stable between cells but we will receive less enzymes in general.
  
  
  - We need to decide how we want to insert the genes to the plasmids, using restriction enzymes or gibson technic.
  
  - Restriction enzymes might cut inside the gene so we need to check all the different sequences that we want to use and maybe even change them a bit so it wont happen. Mosquna thinks that gibson technic might be easier because of that.
  
  - We should check if we can use gibson technic for biobrick creation.
  
  - Safety: Asaf thinks that the personal equipment is very important, so only we use it and nobody else contaminate it.
  
  - Asaf introduced a program called "genius" for planning gene sequences and experiments.
• Everyone.

March 23rd.

March 24th.

March 25th.

• Team Meeting - Assays and Sequences
  We started discussing the sequences and reviewed if they might work in our organisms or not.
  - Assaf spoke with Berta about green light for purchasing materials and she gave it.
  - He says that now there is more pressure on Berta to receive more money.
  - He says we should talk with yonathan about the degradation of the Cl atoms.
  - Assaf wants us to be prepared to plan the cloning for after pesah.
  - We might try to use the igem`s creation for plasmid quantification - https://2017.igem.org/Team:Vilnius-Lithuania.
  - We should contact "genious" and ask if they support iGEM.
  - We should check if anyone tried to syntisys the same enzymes we want to use with some sort of a FLAG.
  
  We should talk to IDT`s representative in israel about the kbs.
  
  How we do cloning:
  
  - Download the annotated sequence.
  - Look for relevant restriction enzymes in the pESC plasmid using the program and mosquna`s notebook
  - We should use 2 resistances for the large enzymatic pathway and another two for all other enzymes we need (like the reductase).
  After we find our FLAG, we should delete the stop codon in our enzyme sequence (if we want to insert it before the flag) or not if we add it after.
  
• Everyone.

March 26th.

• Team Meeting - Rebuilding our Enzyme`s Genes
  Wev'e built gene combinations for our experiments.
  For easier typing' the following groups should be named:
  
  Alpha&beta+223+26=AB
  44a+223+26=44A
  Lip+mnp=Lipp
  
  Suggested groups:
  
  1. Only AB(4)
  2. Only 44A(4)
  3. deHalo+AB(5)
  4. deHalo+44A(5)
  5. CYP+AB(5)
  6. CYP+44A(5)
  7. Only lipp(2)
  8. CYP+lipp(3)
  9. Only CYP(1)
  10. Only deHalo(1)
  
  
  If aiming for redundency:
  
  1. AB+44a(6)
  2. deHalo+AB+44a(7)
  3. CYP+AB+44a(7)
  4. AB+44a+lipp(8)
  5. CYP+AB+44a+lipp(9) -> (!notice: our maximum insert includes 8 genes!).
  6. CYP+deHalo+AB(6)
  7. CYP+deHalo+44A(6)
  
• Everyone.

March 27th.

• Marketing Group Meeting
  Summary:
  - We need to drip some social media sparks (The Facebook page still get weekly viewers without any movement for the past 3 months).
  - Discuss needed presentation files for introducing to the university`s vice president.
  - Discuss companies and organizations that we want to approach.
  - Discuss content of presentations to aid us to present and for sending to others.
  - Plan our next moves and presentation practices.
  - We should Ask Issum in personal about free tickets for Agrivest.
  - Discuss community project management (Each faculty team with different project with a line that combine all of them). A twist is needed for spreading the word in a viral way.
  - Discuss initiation of video concepts.
  - Video Concept - pluralism of Israel's population. Exposing people from the street to genetic engineering and documenting their reaction.
  - We decided to check on other teams and their marketing plan and take good ideas from there.
  - We need to come up with an idea for the script of the animation video.
  - We thought about promoting Bilkis's idea for creating a Regulation system for TCDD manufacturing.
  - We should try and open Instagram page and a Musical.ly page.
  - On the website there should be a tab for stories of people who got injured from exposure to TCDD .
  - And in the same breath - we need to build a website !!
  - About Maintenance of the Facebook page - apparently a rotation between us will be the best.
  - We discussed the fact that it is now the holidays so it`s not a good time to try and approach companies.
  
• Eliya, Lior B, Alon and Sivan.

• Lab Meeting with Assaf, discussing our gene combinatorics.
• Amichai, Lior S, Aliza and Hagit.

March 28th.

March 29th.

March 30th.

March 31st.

April 1st.

April 2nd.

April 3rd.

April 4th.

April 5th.

April 6th.

April 7th.

April 8th.

April 9th.

April 10th.

April 11th.

• Meeting with University vice presidents, Prof. Oron Shagrir and Mr. Yossi Gal.
  
  Oron concluded that he will work with Karen from the international office and will try cover our Jamboree expanses.





• Eliya, Badash and Alon.
  

• Meeting with University vice presidents, Prof. Oron Shagrir and Mr. Yossi Gal.
  
  
  Yosi concluded that because the president of the university will fly to the US next week, we should send him the PPT and one-pager that describe the project and he will try to recruit donations for the project.
• Eliya, Badash and Alon.

April 12th.

April 13th.

April 14th.

April 15th.

• First Lab meeting.
• Everyone.

• Plasmid Restriction
  Restriction in preperation for Gibson Assembly
• Everyone.

April 16th.

April 17th.

• Meeting with Dr. Yonatan Friedman (Microbiology)
• Alon.

April 18th.

• Community project meeting. Deciding on team members that will lead community projects efforts.
• Everyone, Elyia administrated the effors.

April 19th.

April 20th.

April 21th.

April 22nd.

• Gel Analysis of Plasmid Restriction
  
  As can be seen, we got 2 large amounts of fragments in uncut plasmids, and 2 strands in the cut fragments of U, T, H Vectors.
  
  
  
  The L vector, which is outlying (with a single band) was the only vector not restricted with the Cla enzyme.
  To be safe, we decided grow additional plasmids from Glycerol Stock, and preform a Mini Prep and restriction again.
• Amichai.

April 23rd.

• Overnight Cultures for miniprep on 24.4
• Amichai.

April 24th.

• First group meeting in Ein-Karem Campus: Discussing Gene therapy experiment, the option of making a research about TCDD breakdown in the biosphere and recruitment of staff members in Ein Karem Faculty.

  We decided to fuse Ein-Karem students with Givat Ram cell, leaving the option of having a lab experiment in Ein-Karem.

• Alom, Lior and Amichai

• Miniprep and Gel analysis
  
  Bacteria grew well overnight, opaque media in the morning
  
  
  
  
  Miniprep went according to protocol, and gel analysis confirms good results
  
• Amichai.

April 25th.

• Meeting with Danny Porat, V. Dean in Givat Ram Faculty.
• Lea.

April 26th.

April 27th.

April 28th.

April 29th.

April 30th.

May 1st.

• Restriction PCR
  In preparation for Gibson Assembly [GA]
  
• Amichai

May 2nd.

• PCR Purification and Gel Analysis
  
  
  
  
  Bands are as expected, we can move forward with GA
  
• Hagit