Team:TU Munich/Notebook/Meetings

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Contents

Meetings

Tuesday, March 6th

Presentation of Research Results in our internal Wiki

General Remarks

1. Prof. Skerra recommended to stick to a consistent structure when presenting research results.

  • Short summary of what you are about to say (e.g. "AlcR is a simple two component system from aspergilus nidulans which can be induced with ethanol")
  • Give detailed information, such as: nucleotide and/or amino acid sequence, Link to PDB-entry, ...
  • Last but not least, Your personal view & comments

Please make sure to update your entries to match this structure

2. There are some general arguments in favor of our project idea. One of them is, that the application of genetically modified yeast should be legal, because beer is filtered anyway, so no GMOs would be released from the closed system.

Promoters

Ethanol-inducible Promoters

  1. KlADH4-Promoter

The information given in our wiki was presented. The main question discussed was whether this system works specifically (similar to the lac-operon) or unspecifically (similar to a general stress response). If the system works specifically, it is unlikely that the KlADH4 promoter is going to work in S. cerevisiae, because in this case, an unknown specific transcription factor is most likely involved which is not present in s. cerevisiae. If the system works unspecific, we thought that only general stress factors and transcription factors are involved. In this case, chances are that KlADH4 can work in s. cerevisiae, because the necessary factors are most likely present. However, we don't know for sure which mechanism is the correct one (specific or unspecific). Therefore further literature research is necessary for this system.

  1. AlcR-Promoter

The information given in our wiki was presented. The system might be a good candidate, because it seems to be well characterized. Because we do not know many details about this system, further literature research is necessary for this system.

  1. Methanol-Inducible Promoter from Pichia pastoris, and methanol inducible systems from prokarya, such as Methylococcus capsulatis (BATH)

During the discussion about ethanol-inducible systems, two additional suggestions were made. The yeast pichia pastoris is known for its ability to express recombinant proteins upon methanol induction. Maybe this system can be adapted to respond to ethanol - apparently it is based on a specific methanol binding transcription factor. Further literature research is necessary. Also, there is a variety of bacteria which can grow with alcohols (e.g. methanol) as carbon source (Methylococcus capsulatis (BATH)). Maybe they can also provide a system for S. cerevisiae. Further literature research is necessary.

Inducible Yeast Promoters

  1. Chemical Induction: Last year, the iGEM team [British Columbia 2011] created the BioBrick BBa_K517000. This is a Galactose-inducible yeast-promoter which worked for them.
  2. There is a light-switchable promoter system for yeast which has been shown to work for example in a PhD-thesis. See Project Ideas for details. The fact that short light pulses instead of continuous radiation is sufficient to induce expression is an advantage, because light can destroy flavors.

Biosynthesis of small molecules

Resveratrol

There are two approaches for the synthesis of resveratrol, but both require the intermediate Coumaryl-CoA. The 2008 iGEM-Team from [iGEM Rice 2008] tried to submit the resveratrol synthesis pathway in yeast to the registry, but their BioBricks are not functional (for example, they still include forbidden restriction sites). However, the basic functionality of their construct has been shown to work by a different research lab. It would be a good idea to use the DNA they submitted to the registry and make it functional (e.g. removing the forbidden restriction sites...), because this is a great way to improve an already existing BioBrick, which is one of the things iGEM judges really like to see. Volker has already contacted them and got a friendly response.

Xanthohumol

Xanthohumol has never been produced in yeast. However, attempts have been made to alter the hops plant to increase its production of xanthohumol, which shows that beer with increased concentration of xanthohumol is a great idea. Two of the required genes have been well characterized, the other two haven't. The biosynthesis of xanthohumol also includes the intermediate Coumaryl-CoA.


Raspberry Ketone

The synthesis of Raspberry Ketone also includes Coumaryl-CoA.

Caffeine

Caffeine has never been produced in yeast. The required substrate for the synthesis occurs naturally in yeast, because it is a part of the nucleic acid metabolism. Two genes are required for synthesis of caffeine. Caffeine has been shown to inhibit growth of bacteria and yeasts at concentrations similar to that in coffee.

Aspirin

No enzymes are known which produce aspirin.

Nicotine

We decided against the production of nicotine for moral reasons. Our beer should be a healthy one.

Strawberry flavor

Strawberry flavor is not one single substance but a mixture of several hundreds. However, peach-flavor has been produced in yeast already. For interview teams: Ask Prof. Schwab about Furaneol (Dimethylfuraneol)!

Colors and pigments

In addition to the information given in our wiki, please look at [iGem 2011 Uppsala].

Beneficial Peptides and Proteins

The general question concerning Peptides and Proteins is how to ensure their secretion in yeast.

Knottins

Knottins do not fit our requirements well.

Thaumatin

The protein thaumatin is a natural sweetener. Its production should be easy, because only one gene is required.

Lactoglobulin

the digestion of ß-Lactoglobulin exhibits a broad variety of physiological active peptides. How many genes are required?


Discussion & Next steps

Decision on structure of project

We decided to focus on the following modules in our project:

  1. Promoters: Design a library containing three types of promoters: ethanol inducible (for EtOH-Sensor), light switchable and chemically inducible (for control of biosynthesis).
  2. Biosynthesis: The main focus will be on products derived from Coumaryl-CoA, because it is an intermediate for many interesting molecules. In addition to this, we also want to try to establish caffeine-synthesis, because only two genes are required.
  3. Proteins: The secretion of proteins is a good idea, because it will give us a third field of research. This module is promising, because it is likely to lead to easy successes (only one gene required for the production of thaumatin...)

Next Steps

Everybody should assign themselves to one of the following topics:

I) Detailed literature research

For one desired product/construct, find out exactly...

  • How many genes are required?
  • Which genes (including nucleotide sequence)?
  • How do we get the physical DNA (ask a research lab to send it to us, extract from organisms via PCR, synthesis...)?

Please post your results in the wiki

II) "Taskforce Vector Design"

This group should focus on how to transform yeast. Which vectors are available, what techniques are necessary,...? For example, look at the 2011 iGEM Teams John Hopkins and Britsh Columbia. They worked with yeast and got decent results.

Interviews

Interviews with experts will take place on Friday, March 9th. If you are interested in helping, please contact Lara Kuntz.

Miscellaneous

Does anybody know a student from "Brauereiwesen"? It might be good to have one on the team!

Tuesday, March 20th

Overview

The main topics of the meeting were the interviews and the project structure. We agreed that everybody should research his/her topic until the next meeting in two weeks in order to get into the lab as soon as possible (beginning of April). Additionally, we should meet during a weekend (e.g. 15th of April) to work together for a whole day and to address issues such as fundraising and human practice.

We agreed on the following project structure:

Project structure

Promoter and regulation systems

1. Ethanol-inducible

2. Light-switchable

3. Chemically inducible

4. Constitutive active

Biosynthesis systems

1. Coumaryl-pathway

2. Limonene

3. Caffeine

4. Thaumatin

Interviews

1. Dr. Zankow, Beverage Oriented Biotechnology

Security issues

  • In general, brewing beer with genetically modified yeast should not be a problem as far as security is concerned. If we manage to brew a beer with our yeast it should be safe for us to drink it because the filtration process is very efficient. However, we should not let other people who are not part of the team drink the beer.
  • Dr. Zankow remarked that the filtration process might filter out resveratrol and that we should check how much resveratrol is still in the beer after filtration and whether it is functioning.

Alternative Project ideas suggested by Dr. Zankow:

  • Removal of mycotoxins that are found in the grain
  • Beer for a special group of patients (e.g. Celiac disease, Gout)
  • nonalcoholic beer: since nonalcoholic beer does not taste as “satisfying” as normal beer, breweries are searching for something that adds taste to nonalcoholic beer. Dr. Zankow suggested Lactic acid

We are allowed to use the equipment of the chair to brew the beer.

2. Prof. Schwab, Biotechnology of Natural Products

He also made several suggestions for possible syntheses

  • Eugenol (clove-like aroma)
  • Limonene
  • According to him, strawberry aroma is very difficult to make

Prof. Schwab can give use information on several pathways, e.g. for limonene

3. Prof. Hoffmann

  • He was quite critical about resveratrol since only 1 % of the substance is actually functional in the body. Therefore a large amount of resveratrol is necessary to produce a positive effect.

He has mass spectrometers that we are allowed to use, protocols for analyzing beer are available and we can do as many analyses as we want to. Only 5 µl are needed for one analysis.


More detailed information on the interviews are to be posted soon by the interviewers.

Fundraising

  • Lara talked to Prof. Herrmann and he is willing to give us money. We should send a specification of costs to his secretary.
  • Another possible source could be the excellence cluster.

Human Practice

We should use the contacts from the team from last year (e.g. Spiegel). We briefly talked about other institutions that could help us to get some publicity:

  • Carl von Linde Akademie
  • Galileo (as Prosieben is based in Munich)
  • Deutschlandfunk (contact person: Andreas Lang)
  • Quarks&Co (contact person: Ranga Yogeshwar)

TO DO

  • research the topic you have chosen until the next meeting in 2 weeks! (enzymes, sequences, sources etc.)

Wednesday, April 4th

Overview

The main aim of the meeting was to collect all information we gained and to know how we will move on in the laboratory. In some points we are able to order BioBricks already, but nevertheless more information are needed.

People: Volker, Georg, Jeffrey, Mary, Simon, David, Ingmar

Human Resources / General topics

19 Members are in the list (Wiki) and as soon as the semester starts (next meeting) hopefully everyone can come to the meetings.

We will need a brewer -> Georg will ask Hr. Zankhof if some students of his group are interested and Georg will also send an email at the 'Studienkoordination Brauwesen' if some like to join our group. Nevertheless: Does anyone of you know a brewer-student?? Please keep that in mind. Same with Product-&Mediendesign, does anyone knows a student in this field? Will be very helpful in a couple of weeks!

On the Mailing list, everybody of the wiki-list is included (thanks to Fabian) - you already got an email. Fabian will also create an iGem-Emailadress.

Please register at the iGEM-Homepage ! See also the link on the main page (registration on iGEM) After everybody registered, Prof. Skerra will accept us and the team is complete (so no code is needed).

Prof. Skerra ordered an computer for us, it will be in the lab of Prof. Skerra. He also recommends to use the program APE for DNA editing.

On the Saturday we will work together (remember the doodle), Volker will give us a basic introduction of how to check if the BioBricks we like to order for our experiments are okay and working.

Projects

Light-inducible Promoter (Jeffrey)

this project could also act as an independent project (For example if the yeast modification does not work)

BioBricks needed are available -> order them! circuitry (Logic gates) as a model to explain

Chromophore adding instead of producing it (for the first steps) Fusionprotein GAL4+Phyb is available in registry Measuring with FRET

Conclusion: we know everything we need -> ordering BioBricks -> laboratory -> next step: Two persons need to go through the idea with Jeffrey again & help him! -> more data search needed: Sequences where proteins bind


Constitutive Promoters (Georg)

promoters already done in iGEM -> look at team site of those projects behavior of promoters depending on glucose-concentration (the table Georg uploaded in Wiki is important) -> what else is changing during the brewery-process?

Conclusion: asking Prof. Schwab about plasmids with constitutive promoters If constitutive promoters are needed later on -> look in the databases (wiki) or find in registry (Kits available?)


Ethanol-inducible Promoter (Simon)

see details in wiki does not seem difficult -> paper asked Prof. Scherer for different microorganisms

AlcA-promoter in registry available -> BBa_K678001 -> ordering of BioBricks


Coumaryl-CoA (Ingmar)

Pathway from Phenylalanin to Comaryl-CoA does exist! (Prof. Schwab) See interview of Prof. Schwab (David, Volker) -> what could we use from Prof. Schwab? (Roman) -> More data research needed: which products are interesting? story to sell this kind of beer?

Next steps

Next meeting: Mo 16.4.2012 18.00

it is now very important that everybody who likes to participate and likes to bring this project forward will attend at this meeting after the semester break!

in the protocol there are some points on which we have to do more data search - please get involved in these subjects and tell us the results in the wiki.

Monday, April 16th

Organisation

Round of Introductions

Since most of us didn't know each other yet, we did a short round to introduce ourselves. This will hopefully be continued Saturday evening @teambuilding.

Press

jetzt.de contacted us to do an interview about last years iGem project, but as we told them we are participating this year again they will feature this years project as well. Simon did the interview on 17.04.

We showed the 5 ideas for the logo that were also uploaded to the wiki and had a poll on which logo to use as a first design idea:

Logo I Logo II Logo III Logo IV Logo V
9 Votes 1 Vote 0 Votes 0 Votes 12 Votes

Final decision was to work on Logo V.

Additionally we decided that Jeffs Idea runs out of concurrence and could be used for the wiki design.

Vector Graphics will be created by Dennis

  • Maybe someone knows a Mediendesign student who would like to join the team ?

Ideas for the logo design:

  • keep it simple
  • banderole thinner
  • outer oval in bavarian blue/white pattern
  • use the weihenstephaner logo for the upper part of the bottle

Comments by Prof. Skerra

  • Sponsoring Letter
    • Sell the beer brewing idea more as a working example than as the main idea to make the project more generally acceptable
    • Prof. Skerra improved some parts of the sponsoring letters and is open to give more advice
  • Work on the website
    • Put the group photo online
    • Add more information

Research

Prof. Schwab

Send a mail to Roman with the names of the enzymes that we need (trivial name is sufficient)

We also thought about the option to implement more of his ideas but rejected this notion since we already have sufficient nice ideas to work on.

Coumaryl

List of required genes to Roman. Please copy-paste Information to the new page on the wiki!

Limonene

Mail to Prof. Schwab: Is the one enzyme that we suppose to be sufficient really enough?

Caffeine

2-3 Enzymes (we will use the pathway that uses 3 since it seems more realistic to work) Roman contacted the authors of some papers but did not get any response from them so we do not know how we will obtain the genes.

Gene synthesis is possible (3k bp) but it is questionable whether it is really worth the effort since pathway is rather complicated and concentrations will most likely not reach physiologically active concentrations.

Thaumatin

2 other sweet substances are already available as biobricks but their potency is questionable.

The gene Thaumatin is, as most of the other genes, not BBRFC10 compatible but this does not really pose a problem since it is easily fixable by quickchange mutagenesis.

Gene Synthesis

It is probably very time efficient to synthesize some genes, therefore we need to check what is the cheapest/fastest solution here. Look at

  • Mr Gene : http://biomatik.com/CustomServices/CustomGene/Gene-Synthesis.aspx
  • Check the iGEM Sponsors : https://2012.igem.org/Sponsors ?
  • Last years team : Mr Gene as far as I can tell (Fabian)

Promoters

Simon will begin working on the KlADH4 system next week and use a chemical inducible promoter for the required components.


Saturday

We will meet Saturday 21.4.2012 at 14:00 at the Seminar room to do an in-depth research afternoon.

Agenda idea:

  • Find Research/Lab Groups
  • Tour through the lab
  • Introduction on what to pay attention when working with BioBricks
  • Research in smaller groups

BRING LAPTOPS!

Afterwards we will eat something and have a beer!

Saturday, April 21th

Introduction by Volker into BioBicks and Methods

Quick introduction by Volker on BioBrick RFC10 & RFC25, Codon usage

Team forming

We split up into groups to do research in smaller groups. these groups will also realize the sub project so everybody who was not present please select a group an check with the group-leader.

We met in these smaller groups and only report our progress in the main meetings.

Next Meeting

  • The time for the next meetings will be Tuesday 20:00. next date for the meeting will be May 1rst.
  • Ideas
    • Human Practice
      • We need to come up with something new and fancy
      • Interviews : not new
      • Panel Diskussion : very time consuming
      • TV - Galileo : we need to check
      • Create a blog : last year HP price was awarded for a blog
    • Partsregistry RFC
      • Machine Readable Entries
      • Standardized Information
      • Check with Randy
      • Usage maps
  • Research
    • We need to accumulate data on sequences so we are able to order them and primers to make them RFC10/RFC25 compatible
    • Please add the following table to all sequences:
Name Length RFC10 RFC25 Codon Usage NCBI
... ... bp NxSite(location) ... NxSite(location) ... use http://gcua.schoedl.de/ and check whether we need to optimise NCBI nucleotide link
  • Tell Volker so he can design & order Primers.
  • Teambuilding at Huber in Freising

Tuesday, May 1st

General

To Do List

  • The photo of our group ought to be placed on our website: [http://www.igem2012.de| iGEM 2012]
  • We should have a small team, which cares about the formulation of abstracts and project descriptions concerning our experiments and ideas in order to put that on our website
  • The formulated sponsoring- letters should be uploaded in our wiki, to make them available for everyone
  • @ Sponsoring- Team: When do you send out the letters?
  • At the next meeting (15.5.2012, 20:00): Skype- Conversation with the student of Stuttgart (who wants to refer to us in her masters thesis)
  • Prof. Skerra will be contacted concerning the following issues:
    • general health and safety briefing
    • outstanding team- applications (at the official iGEM Website)
    • getting contact to people who have experience in working with yeast (especially in transformation with multiple genes, genome integration and heterologous expression) and would give as a "crash course"
  • Contact Prof. Schwab on Monday, May 7th, after his lecture "metabolic engineering and production of natural compounds" (also for getting a "crash course" / good literature concerning yeast etc.)
  • everyone of us should acquire general knowledge concerning transcription and translation in yeast (e.g. ribosome binding site, etc.)
  • David should get a list with required chemicals and other stuff, we will need / perhaps need in the lab (for our sponsors)
  • If anyone of us has / had personal contacts to enterprises in the field of biochemistry/ molecular biology etc., please tell David
  • Get contact to the foundation that supported/ supports Team Heidelberg
  • @ Volker: Can you put some general information (e.g. concerning primer- design) in our wiki?(e.g. regarding primer- design, etc.)
  • Each team should make a list of the required BioBricks, sequences (to be synthesized - eventually codon optimized and with standard compatibility), and primers (sequences one wants to amplify, respectively, that primers can be designed) and give it to Volker (further required BioBricks can be put on the existing list ([http://igem.wzw.tum.de/index.php/List_of_BioBricks_to_order click here]) in our wiki, respectively) until Friday, May 4th., for the order of the BioBricks should be arranged in the next days

Sub- Team Reports

Human Practice / Press / Video

(Jara)

  • List of appropriate magazines, which could report on us:
    • SNIP- Magazine (the chief editor happens to be my (Roman) cohabitant, so i could care about that)
    • Zeit Wissen and Zeit Campus
    • Freisinger Tagblatt
    • Biospectrum
    • Transcript
    • Efellows
    • after a few weeks and results (hopefully): Sueddeutsche Zeitung (again)
    • Galileo
    • if there are other ideas, tell Jara (e.g. SpiegelOnline, PM)
  • Video
    • Jara had the idea to make a video that looks like a beer- advertisement (bored people sitting in a beer garden, super yeast comes, giving them the new beer and happy end ... )
    • to realize this, we could contact the Filmhochschule München

Ethanol inducible promoter

(Simon)

  • Simon starts with this part of our project on May, 2nd.
    • For now: simple yeast transformation experiment with GFP and pYES2 als vector system
    • Later: Amplification of a special promoter out of Kluyveromyces lactis

Light-inducible [romoter

(Jeff)

  • A PhD student will be contacted, in order to get some sequences
  • As described in the project description, this group wants to change the Gal4BD for LexA

Limonene

(Lara)

  • Lara could start in about 2 weeks with the labwork
  • The sequence is available at the chair of biotechnology of natural compounds (Prof. Schwab), but a few codons should be optimized. Besides it should be checked, if the sequence is complete (because the comparison of the available vector and the sequence on NCBI have not the same length (perhaps due to a his- tag))

Caffeine

(Roman)

  • Sequences can only be received by gene- synthesis
  • Sequences have to be codon optimzed and partially mutated due to forbidden restriction sites
  • Only the coding sequences will be used for amplification
  • This project will start later

Thaumatin

(Jonas)

  • This projects lab work can start soon, but since Jonas did not participate in the last meeting, further information will come at the next meeting

Next Meeting

  • We decided to continue the "main meeting" in a two weeks interval and determined May, 15th 20:00

Tuesday, May 15th

The first draft of the logo was edited. The letters should be written in “altdeutsch” to enhance the Bavarian effect. Another concept was introduced: “tumb”eer with labels for the bottle front, back and the top. The different flavors/ingredients (caffeine, limonene, etc.) were symbolized by different colors. The idea was to present a beer in the future which is created by the consumer. It could be presented for the iGEM competition as a view for 2050. However, the team-logo still needs to be designed.

Homepage

Martin will continue to work on the homepage and add the team picture.

Report by Roman

  • People of the institute of professor Schwab will participate in our team as advisors. They will receive accession to the Wiki and officially be registered for the iGEM ream.
  • Maybe the new advisors can offer a crash course in yeast-techniques. Complexity still needs to be defined. The most important issue would be the organization of yeast gene structures and the methods of selection. The genome integration and afterwards the selection is another important topic.

Team members need to read papers about gene co-expression in yeast!

Time is running out!

  • Bricking needs to be done soon, because it takes a lot of time.
  • Thaumatin, limonene and coumaryl could start very soon (1-2 weeks).
  • Caffeine needs to be synthesized.

Sponsoring

Many different sponsors are acquired. Most important is Eurofins, because they will synthesize oligonucleotides for 0,10 Euro per base. More information are in the sponsoring section

Gene synthesis

  • 3 genes need to be synthesized for the caffeine team
  • 2 genes might be needed for the thaumatin team (they might be able to get the genes from Heidelberg)
  • 1 gene is necessary for the light-inducible promoter team

Final orders need to be posted in the sponsoring section

Lab work

  • Who has time? Timetable in internal wiki!
  • A clear defined agenda of your lab work should be made to show it Prof. Skerra. This is very important for your lab work, too!
  • Genes need to be ordered now or very soon. Otherwise we will run out of time!

Presentation of thaumatin

The presentation needs to be uploaded for everybody.

Introduction of the iGEM team to the institute

Idea: BBQ-night

BMBF

The invitation to Berlin was discussed and afterwards Martin, Lara and Volker were drawn to participate.

Public relations

Laborwelt will post news of our team on their website and in their newsletter. Possibly in the end of our project an article in their magazine will be published. Weekly updates, links and pictures are necessary for them.

Thursday, May 24th

Organisational Issues

  • Team Instructors: Doreen Schiller (from Prof. Schwab's Department) already registered as an official team instructor for us. others may follow. Thanks to Roman for taking care of this!
  • Trip to Berlin: Fabian, Lara and Volker have been signed up to represent our team and present our project in Berlin on June 28th.
  • Barbecue: We will have a BBQ on June 5th, at 18:00 o'clock. Jara is the main contact for this. She should also get in touch with Klaus Wachinger to organize "Bierbänke" and everything else we need.
  • Our homepage www.igem2012.de should be updated. We also need a logo
    • Prof. Skerra made the following suggestions for the "Advertisement-Design" which Dennis presented previously (beer bottles): it should be "Alc 5.2" instead of "Alc 5,2, the name should be changed from "tumb" to "TUMbrew", the color of "glowing in the dark" should be changed to something greenish and this design (glowing in the dark) could/should be used as a first logo/picture on our homepage.
  • There will be a list with our inventory (excel-sheet)

Project

General Comments Prof. Skerra

  • The content is more important than the compatibility with iGEM standards
  • Don't forget regulatory elements
  • Find our about N-End-Rule
  • We should make a brainstorming seminar, where we discuss functional examples from the literature about co-expression of heterologous genes in yeast. When looking for DNA-Sequences, it also helps to make a "patent research", for example using the homepage of the EPA (europ. Patentamt), because in patents, the DNA-Sequences have to be shown!
  • In DNA sequences, the following unusual letters are used:
    • n = any nucleotide
    • y = pyrimidine (C, T)
    • r = purin (G, A)
  • When showing DNA-Sequences & designed primers, it is best to show two sequences (preferably a WORD-Document): One that shows what the final construct should look like and another one that shows the template DNA (usually a plasmid) and the primers you designed for the PCR. Also, you should know exactly which restriction enzymes you want to use for the cloning of your parts and why. Also have the GeneBank or Uniprot or PDB identifiers (codes) ready, so that you can easily show the respective enzymes.

Sub-Projects

Caffeine
  • Make one long, functional gene-synthesis that contains everything: the genes coding for all three enzymes, regulatory elements and restriction sites to allow for easy excision of each gene and cloning into plasmids.
Limonene

We have two sources for this:

  • A plasmid from Prof. Schwab. PCR will have to be done with this. This is lavendula limonene synthase
  • A Biobrick on the distribution plate (lemon limonene synthetase). Unfortunately, this part already contains an E. coli RBS, so we will also have to perform a PCR with this part.


Tuesday, May 29th

"no protocol"

Tuesday, June 5th

Primer design

  • 3 enzymes were accepted by Prof. Skerra and could be ordered (phenylalanine ammonia-lyase, limonene synthase, ???). We decided to order one primer pair with the consensus sequence and another without to prove whether the consensus sequence is necessary or not.
  • For those who have to design primers: the region that anneals to the gene should be 18 bases long and should contain 10 G/C’s. Furthermore the overhang should be 6 bases instead of only 5.
  • Everybody who designs a primer should consider how to detect the enzyme: enzyme assay, antibody detection (abcam is a supplier of antibodies,you can look there for an appropriate antibody! A search engine exists as well…does anybody know the name???) or detection via tag (strep-tag or his-tag)

Primer purification

  • Primer that are longer than 60 bases should be purified

Co-expression of multiple genes in yeast

  • We need detailed information over multicistronic constructs in yeast! How is it done in other labs??? Look for papers and create a word document with sequence information and annotations.

Restriction sites

  • Restrictions sites inside a gene will be removed by quick change PCR after we proved the proper functionality

Lifetime of proteins

  • Look for the N-end rule.

Lab work

  • Saskia, Andrea, Lara and Daniela start working in the lab next week

Recruitment of a chemistry student to the team

  • Some enzymes may be detected via assays. Therefore we need different chemicals which may be produced by chemistry students during a practical course
  • Knowledge expansion of out team
  • Ingmar tries to recruit a chemistry student

Presentation of Jessica

this report is incomplete

Tuesday, June 12th

Introduction of new approach for vector design by Volker

  • multiple cloning site is modified before cloning to fulfill iGEM standards
  • lab work aiming vector improvement starts June 13th (Saskia, Daniela)
  • primers have to be redesigned to match the new standard and have to be ordered AS SOON AS POSSIBLE

Planning of lab work

  • Quick change mutagenesis will be used to eliminate restriction sites that do not match iGEM standard
  • Meeting for lab members that will start their work this week: June 13th, 3 pm
  • Some groups got their primers approved by Prof. Skerra (Coumaryl), some need further planning (Thaumatin, Light-Inducible Promoter)

Primer design

  • Everyone should use the same stop codon (TAA)
  • 6 bases should be added to the primer ends (not GCGCG, but rather another restriction site)

N-end rule

  • Groups should consider the N-end rule while planning their primers
  • If protein contains an glutamic acid or similar amino acid at the N-terminus, a small amino acid like alanine could be introduced N-terminally
    • this might lead to increase in protein stability!
    • check with information provided by Ingmar on the main page

Codon optimization

  • We have to look up a table containing codons preferred by yeast
    • Uli Binder (employee of Prof. Skerra) can be contacted concerning codon optimization
    • Also remember the online tool GCUA (Graphical Codon Usage Analyser) which can be found here: http://gcua.schoedl.de/

Lab book

  • Discussion about whether lab book should be digitally or handwritten
    • lab team that starts this week will use an ordinary lab book (one book each)
    • digitalisation must be discussed in next meeting!

Brewing process

  • our yeast strain is a top-fermented yeast. Top-fermented yeasts allow for fast brewing. BUT: Does our yeast grow in Gyle medium that is used in the brewing process?
  • our two new team members (brewing students) will contact Simon concerning the yeast strain and will start an experiment to get information about growth behavior of "our" yeast in Gyle medium
  • other things that need to be discussed:
    • Is a specialized brewing yeast strain available that is fully sequenced?
    • How can we make sure that the beer will be sterile and free of genetically engineered yeast?
    • pasteurisation does kill the yeast but does not eliminate the dead yeast cells
    • filtering might be a suitable approach, but oxygen will be lost and would have to be introduced afterwards
    • brewing students will contact a specialist of our university concerning filter techniques
    • it has to be checked whether the "Gentechnikgesetz" allows for dead genetically modified organisms or not 
this report is possibly incomplete

Tuesday, June 19th

Present: Katrin, Dennis, Ingmar, Daniela, David, Volker, Jonas, Martin, Fabian , Jara, Mary, Simon, Jeff, Georg

Missing: Alois, Andrea, Lara, Nadine, Roman, Saskia


Lab-Reports: Simon, Daniela & Saskia, Lara & Andrea

  • Simon: Vector with ethanol promoter is finished. Problem is negative control.
  • Daniela & Saskia : igem compatibility of pYES, sequencing will be done tomorrow.
    • one of the forbidden restriction sites is located in the 2 mu origin
    • is there an equivalent to mini-prep? --> Ingmar
  • Lara & Andrea : Primer arrived, plasmid from Prof. Schwab are prepped, plasmid from iGem plate will be prepped tomorrow.

Ordering of Primers

  • Which primers have been ordered and which ones still have to be ordered?
    • All Primers that are in the wiki are already ordered
    • Jonas Primer are finished, he will check with Volker and then order
    • Some bad primers were ordered

Inventory

  • The person who orders stuff is responsible for it.
  • If you get a new tube, enter it in the inventory list
  • Names in the wiki and on the tubes must be the same.

Ordering of BioBricks

  • Is the list complete? --> Nadine will order the bricks tomorrow.
    • Constitutive promoters are missing

Planning of Labwork

  • Who is available to start with PCRs, cloning of constructs, ... ?
    • Time Schedule is on the main page, Fabian will contact people to fill in information
    • Who wants to test if our yeast is suitable for brewing?
      • Martin will contact brewers about sequenced brewing yeasts and where we get the yeast
      • David will brew next week and he will give us some gyle

Discussions

  • Promoter-System: Which ones will be used for characterisation? Which ones will be used for brewing?
    • iGEM Kit already contains one constitutive promoter (sequencing good) and an adh1 promoter (sequencing inconsistent)
    • Creating a promoter library with characterised strength.
      • promoters: adh1, tef1, promoter kit, 3x mcyc
      • is galactose contained in gyle? -> Martin.
    • Co-Expression of genes: Did anyone find out anything?
      • For us question is solved: promoter + gene + promoter + gene etc.
      • Caffeine: Complete construct will be integrated into the genome at once (zack!)
  • Timeline:
    • Will be added later
      • Eligible for genome integration: limonene, thaumatin


  • Berlin: Poster, discussion about our opinion about GMOs in food
    • Poster
      • Everybody should helpt
      • Coumaryl CoA : Pathway Bild
      • Top: TU + Logos + Title
      • Introduction : about 1/4 of space, circa 200 words
      • Group picture
      • Sponsors
      • Every Team 5 sentences + 3D-Struktures-> more pdb/pymol files?
      • Deadline Thursday
    • GMO
      • -> Brauerfragen


  • Sponsoring / PR: Booking of Hotels for Amsterdam, Homepage. Any other news?
    • We need a cost listing (Hotel/Journey)
    • Website needs to be polished.
      • Martin will take care.
      • Dennis has pictures
      • Use abstracts from poster
    • CL Prosieben
    • Exzellenzinitiative
    • Dirndl Picture
    • Mainly Galileo

Tuesday, July 3rd

People: Simon, Jeff, Saskia, Daniela, Nadine, Alois, Martin, Ingmar, Katrin, Mary, Lara, Volker, Roman, Fabian, David, Georg, Jara, Andrea

Missing: Dennis (ill), Jonas

Time: 20:00


Introduction

(Mary)

  • Our goal is Boston, project plans should help us getting us there. (Mary)
  • Project plans are necessary to communicate who is doing what and synchronise work. (Simon)


Lab-journal + Rules

(Mary + Simon)

  • Project coloring
  • One big Labbook
  • write an aim of experiment
  • Fabian will send a guide on how to name pictures
  • Gown is not always necessary, but please wear an igem badge
  • tomorrow: update the inventory list
  • is it okay to do an meeting agenda + documentation --> general yes
    • send ideas for meeting agenda to mary/simon or directly edit in the wiki


Lab-Reports + Project Plans

(Teamleader)

  • place project plans in the dropbox

Coumaryl

  • Cloning should be done next monday
  • One enzyme will be synthesized
  • Expression will be done in E. coli to test functionality, yeast is slower
    • Characterization in yeast should be done asap
    • Expression in possible problems in E. coli due to disulphide bridges
  • Quickchanges will be done later, focus is on results
    • Substrates for essays could be available at Schwab lab
  • Purification manual in the pYES2 kit / ask Doreen
  • Characterization and quickchanges should be done in parallel as purification can easily take over 2 weeks
  • Order primers! (Ingmar will do it)


Limonene

  • 2 different enzymes
    • BioBrick: pcr done (add strep tag / RFC25 + removal of rbs)
      • ligation should start on monday
    • Schwab: miniprep did not work
      • ligation should start mid next week
    • Charactisation with gas-chromatographics (-> Stefan Gilch)/strep tag


Constitutive Promoters

  • BioBricks have some errors -> genome PCR is necessary
  • Expression will be done in BioBrick vector
    • pYES2 could be used as iGEM compatible shuttle vector without promoter, but removal of promoter + terminator is necessary
  • bad/none sequencing does not necessarily mean the brick will not work
  • primers will be orderded until Friday
    • primers need to be blasted against the whole genome
  • promoters should be ready in 2 weeks


Light-Inducible Promoter

  • Limiting factors: ordering of BioBricks + iGEM compatibility of pYES2
    • Bricks should arrive soon
  • will need constitutive promoters later on
    • for purification of phycocyanobilin some algae are needed
  • assembly should be done two weeks after arrival of all necessary parts


Thaumatin

  • Primers were not ordered due to miscommunications
    • eventuality not bad since synthesizing the gene makes more sense than ordering
  • this group can help other groups (or test mass-spectrometrics + brewing tests)
  • Jonas is missing - is he still in the team?


Caffeine

  • Promoters can be reduced in length but become more more ineffective
    • Annotation is bad
  • Synthesizing 9k bp takes very long and will be very expensive
    • Prof. Skerra himself is possibly against the long sequence
    • Plan is to synthesize all parts separately
      • clone separately/together
  • second AS after start codon is hard to be identified in 3D structure (Volker can help)
    • construct should be codon optimized / rna loop avoided


Vector Design

  • 4 quickchanges were successful
  • 1 Ala needs to be inserted
  • should be done on Friday
  • results should be available on Monday


Genome Integration

  • iGEM vector will cause some loss


Milestones

  • Expectations of last milestones were not met.
  • What do we want to realize until the next meeting?


Report: Brauerfragen + Experiments

(David H., Martin)

  • Sequenced (needed for genome integration) brewing yeast apparently not available (Ingmar will send a paper to Martin)


Report: Berlin

(Lara, Volker, Fabian)

  • Frankfurt is also doing an artificial sweetener in yeast
  • Interview with Mr. Laqua from Laborwelt
  • Common Project with all German Teams on August 25th


Poster for CAS-Conference

(Fabian)

  • Fabian will contact Dennis for the templates
  • Volker will do the abstract


Idea: Meet with politicians - Human Practice

(Volker/Fabian)

  • Contact politicians to inform about and discuss our project
  • Cooperation together with Laborwelt
    • Meet with experts and reflect (not new)
    • Inform politicians about biotechnology/synthetic biology (new) -~ discussion etc, Jara will make a concept


Film Making

  • Jara knows a student who will make a commercial with at the beginning of august
  • Mail to Prosieben was sent, waiting for a response


Idea: Invite a school class to do an experiment

(Nadine)

  • Nadine will determine possible 2-3 experiments


Prof Skerra wants a list of everything what we need

  • every group should send a list of required things to David


Minimum requirements to go to Amsterdam/Boston?

  • Discussed next meeting.

Next meeting (Wed. 9.00) + milestones

Tuesday, July 10th

Present: Jeff, Alois, Andrea, Jara, Daniela, Nadine, Simon, Lara, Mary, Ingmar, Fabi, Martin, Georg

Missing: Volker, Roman, Dennis, Saskia (Freund Operation), Jonas, Katrin


Report: Lab + Milestones

(all)

    • Biobricks arrived
      • Simon will plate out tonight
    • pYES2
      • site restricted mutagenesis is finished
      • should work in bacteria, possibly not working in yeast due to mutagenesis in mu2 ori
      • please don't use up all of the vector in tube P50
    • Limonene
      • PCR finished
      • Ag1 is missing
      • can someone do the miniprep on saturday? (Alois/Martin)
      • Milestones:
        • until monday next week: tranformation in yeast characterisation
        • monday in 2 week: start characterisation
          • strep tag -> schwab gcms analysis of products, ask schwab/gilch (substrates)
    • pSB1C3 RFC25 compatible (assigned to jeff)
      • Volker will take care of the RFC25 compatible pSB1C3 (Potsdam/Freiburg or make on our own)
      • remove rfc25 from pYES2 with rfc10 restriction sites and ligate into pSB1C3?
        • Jeff already did this with one part (->BBa_K105005 see Labbook)
      • bricks with streptag cannot have this tag removed in pSB1C3
    • Coumaryl
      • Ligation can start tomorrow
      • one PCR has to be repeated again
      • characterisation should start next week
        • needs certain educts which are either not available or very expensive
          • ask people from papers/ prof schwab
          • subsequent pruduction of educts is not realistic
    • Thaumatin/Caffeine
      • synthesesis is not ordered yet, they asked whether we want the synthesis in a pUC vector (better for >1kbp), should be ordered tomorrow
        • should arrive in 10 days
    • constitutive Promoter
      • digestion of bricks is done
      • georg vaccinated his bricks
      • adh promoter is correct
      • skerra was not pleased with presentation
    • light switchable promoter
      • purification of phycocyanobilin started -> methanolyse in progress
        • should be done on friday
      • jeff will start cloning tomorrow
  • Teamleitermeeting (Mary)
    • What should we present in Amsterdam (Brewing with Limonene is realistic, but with Coumaryl probably not possible)
    • Tuesday 17:30 (tell Roman/Volker)
  • Chaos
    • If you are not sure how/what to do, ask before doing stupid things
  • Discussion: Do we want minimum requirements to go to Amsterdam/Boston and what should they be
    • hard to differentiate between people who are working in the lab and outside -> number of days is possibly not a good idea
    • Attendance in Meetings (80%)
      • excuse is necessary in advance nonetheless (for those 20%)
        • note it in the wiki below the agenda
    • We will pay more attention to presence in lab
      • We will give a warning if people are not sufficiently present
  • Report: Brewing Questions (sequenced Yeast - potent?, experiments, ...?) (Martin, Mary)
    • only genome shotgunning, no whole genome sequence
    • we will test whether our lab strain will survive (-> Martin/Alois)
      • hopefully their only disadvantage is that they will produce beer slower.
  • Introduction into Yeast Transformation / Proteinexpression in Yeast (Simon, Katrin)
    • add protocols to dropbox
    • contact simon as soon as you start doing something
  • Report: Kostenaufstellung und Substanzenliste für Enzymassays (all/David)
    • Just do it, send it to David asap.
  • School visit (24.07.12) - who helps Nadine? (Nadine)
    • Jara will help
    • Nadine will check what substance are needed and ask skerra/school
  • Homepage - who takes care about it? (Martin)
    • Jara / Martin
  • Poster for CAS (Fabian)
    • PDF will be placed in dropbox for criticism
  • Human practise: politics and movie (Jara)
    • politics
      • FDP guy is interested and will bring friends
      • ask skerra to be expert / ask him for date
      • will take place mid september
      • ask for hoersaal 16
      • ask braufaesschen group / braufaesschen prof
    • movies
      • jara will do script
      • mid august

Tuesday, July 17th - Meeting of group leaders

Tuesday, July 17th

Tuesday, July 24th

Tuesday, July 31st

Tuesday, August 7th

Tuesday, August 14th

Tuesday, August 21th

Retrieved from "http://2012.igem.org/Team:TU_Munich/Notebook/Meetings"