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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

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.

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.

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

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.
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:

Promoters: Design a library containing three types of promoters: ethanol inducible (for EtOH-Sensor), light switchable and chemically inducible (for control of biosynthesis).
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.
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.

Logo

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

Team logo

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

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 yeast, possibly not working in bacteria 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 mini-prep on Saturday? (Alois/Martin)
Milestones:
- until Monday next week: transformation in yeast characterization
- Monday in 2 week: start characterization
  - 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 strep-tag cannot have this tag removed in pSB1C3

Coumaryl

Ligation can start tomorrow
One PCR has to be repeated again
Characterization should start next week
- Needs certain educts which are either not available or very expensive
  - Ask people from papers/ Prof. Schwab
  - Subsequent production of educts is not realistic

Thaumatin/Caffeine

Synthesis 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 Promoters

Digestion of bricks is done
Georg vaccinated his bricks
ADH promoter is correct
- Prof. Skerra was not pleased with presentation

Light-Inducible Promoter

purification of phycocyanobilin started -> methanolyse in progress
- Should be done on Friday
Jeff will start cloning tomorrow

Team Leader Meeting

(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 is available, 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: Cost listing and list of substances for enzyme assays

(all/David)

Just do it, send it to David asap.

School visit (24.07.12)

(Nadine)

who helps Nadine?
Jara will help
Nadine will check what substance are needed and ask Prof. Skerra/school

Homepage

who takes care about it? (Martin)
- Jara / Martin

Poster for CAS

(Fabian)

PDF will be placed in dropbox for criticism

Human Practice : politics and movie

(Jara)

Politics:
- FDP guy is interested and will bring friends
- Ask Prof. Skerra to be expert / ask him for date
- Will take place mid september
- Ask for hoersaal 16
- Ask Braufaesschen group / Braufaesschen prof
Movie
- Jara will do script
- mid august

Tuesday July 17th - Meeting of group leaders

Attending: Simon, Mary, Volker, Georg, Jeff, Lara, Roman, Fabian, Dennis, Martin; Missing: Jonas

Prerequisites for medals

(Simon)

Bronze Medal

team registration
team wiki
poster and presentation at regional jamboree
new submitted and well characterized part (already accomplished)

Silver medal

functional BioBrick with registered function (should be quite easy)

Gold medal

At least one of the following criteria has to be achieved:

improvement of an existing BioBrick (limonene- team: RFC10 ==> RFC25)
help another team (e.g. cooperation with the team of Frankfurt?)
human practice

Theoretically we should get the gold medal. However, a gold medal does not necessarily lead into the final. It is important to focus on exact characterization and to submit it to the registry!

Aim: Special price?

To win a special price, the team has to come up with something special! Examples (i.a.):

Best measurement approach

E.g. cheap and easy methods for characterization

Best modelling

Best new biobrick (natural)

This biobrick should be something really crazy...

Best new biobrick (engineered)

E.g. light switchable promoter system. Each team leader is to think about his project - if there is potential to win one of the special prices, we can all help this team!

Best new standard

This does not necessarily mean the classical standards. It is also possible to focus on an improvement of existing procedures or of the interface of the registry, to make it more user-friendly (Volker has ideas)

Best poster

Best presentation

Best wiki

Last year's feedbacks

Rethink your characterizing experiments precisely (assays, etc.), think of positive and negative controls, etc. Ask Prof. Skerra or our advisers, when it comes to the characterization of your enzymes!
Application? Did you finish your project? That is to say, that it would be great if had brewed a beer at the end
Modelling: One negative thing last year was the fact, that the modelling based on literature data. We should link the modelling with the lab- work this year, so ask Fabian what data he will need.
Crowd founding: Platform already exists - we could create an own iGEM sub-folder
Track choice: There are several tracks and we have to choose one favored track (e.g. "food and energy") and a second one (which one?)

iGEM and Prof. Skerra

Run the fridge in the lab - already accomplished
Problem with lab work in the evening: There has to be present at least one member of the chair. Works on the computer are ok

Team status

Light-Switchable Promoter

Jeff has a lot of exams in the next days, he needs about 3 weeks. There were also some problems with "fake"- BioBricks of Harvard

Caffeine

Waiting for the gene synthesis. If received, it is realistic to proceed to the characterization

Thaumatin

Problems with the purification (strep tag is not possible due to secretion)

Limonene

Problems with remaining restriction sites

Coumaryl

There will be problems with the characterization, due to expensive substrates and the amount of enzymes.

The "big projects" will probably not exceed the expression, but there are smaller ones, which could

Important things to do

Concerning the brewers: reserve the equipment for limonene beer and thaumatin beer
Contact Georg concerning the required promoters
Make a yeast transformation with the integration vector (contains mOrange)

Tuesday July 17th

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

Missing: Jonas

Report Team Leader meeting

(teamleaders)

See previous section

Prof. Skerra about working times

Presence of one person from the institute is required for any work in the lab (Volker is external!)
If the fridge in our lab is working we can use it!

Explanation: Modelling

(Fabian)

See Modeling Section for details

Characterization

(Fabian)

Generally pay attention to catch essential time-resolved characteristics of the behavior (better resolution where interesting things happen)
Fabian will be in the Lab from August 10, so ask before you start your experiment (possibly not on the same day)
Investigate whether mRNA characterization is possible (probably only interesting for promoters qPCR)
Make an appointment with Prof. Hoffman to ask what is possible first week of August (Nadine)
Create list of question in the iGEM dropbox until next Tuesday.

Explanation: Promoters

(Georg)

See Constitutive Promoter for details

Lab Report + New milestones

(all)

pYES2

DONE!
we still need to check functionality

Limonene

Citrus (BioBrick): ligation did not work (transformation should start Monday/Tuesday next week)
- problem should be unrelated to pYES2
Lavender (Schwab): overlooked restriction site, need one more quickchange
- Monday in 2 weeks: start characterization (one week delay)

pSB1C3 RFC25

-> BBa_K365005

Coumaryl

One week behind (ligation did not work, transformation should start tomorrow/2 days)
PCR worked
All enzymes should be done in pSB1C3
Enzymes in pYES2 beginning of next week
Characterization should start next week

Thaumatin/Caffeine

Communication troubles with synthesis, ordered today --> 10-18 days

Constitutive Promoters

Cloning in progress

Light Switchable Promoter

purification DONE!
cloning needs help!
could be finished in approximately 3 weeks if all works well

Trouble in the Lab

Marking a tube only with number is not sufficient, please put a sticker on it with description
Can we somehow arrange sequencing outside the chair ?

Report: Yeast survival in gyle

(Martin / Alois)

In progress

Replace tum brew - synbio beer

maybe print stickers ?

Report: Cost listing and travel

(David)

Talk to david, do not contact external people directly
12,000 possibly available from Weihenstephan, meeting mid-August
David needs the list, check back with Prof. Skerra before presentation
Discussion about the journey to Amsterdam - does everybody like to stay?
- create a doodle when you are available
don't forget to get passports in time!

Report Genome Integration

(Martin/Alois)

postponed
Pay attention to reregulations during the brewing process

Tuesday July 24th

Present: Mary, Simon, Roman, Jeff, Saskia, Jara, Daniela, Katrin, Andrea, David, Marthin, Nadine, Georg, Ingmar, Dennis

Missing: Fabian (@CAS conference + iGEM meetup afterwards), Lara (out of town; I'll be back in lab on Thursday), Alois (out of town), Volker

who writes protocol? --> Ingmar writes protocol

Lab Report + New milestones

(all)

Coumaryl

5 BioBricks have been created (site directed mutation and sequencing still necessary).
New Milestone: Successful transformation in yeast.
David will contact Andreas Reichert for external sequencing.

Limonene

Limonene synthase sequences are successfully cloned in pSB1C3(site directed mutation and sequencing still necessary).
New Milestone: Successful Ligation in pYes2 RFC 25 and pSB1C3 of both Limonensynthase sequences.

Caffeine

Waiting for gene synthesis.
New Milestone: Start of cloning in pYes2 and pSB1C3.

Light Switchable Promoter

Ligation in yeast2hybrid vector failed-->repetition.
New Milestone: depends on whos working for Jeff.

Constitutive Promoters:

waiting on meeting with Prof. Skerra to order primers.
Wich system should be used to test the promoter strength?
- One enzyme essay and one antibody assay should be possible.
New Milestone: Test the activity of TPI on ethanol dependency.

Report Genome Integration

(Martin/Alois)

Report Yeast survival in gyle

(Martin / Alois)

pYES2 - quick introduction

(Ingmar)

Organization

Meetings at 18.00 ?
- next meetings will take place at 18:00- 21:00h.
When to go Amsterdam (doodle) ?
Jonas has left team.

Human practice

Interview with Prof. Hofmann (Nadine) & Prof. Schwab (Lara)
- only one question???!! No progress!
- Lara, if Prof. Schwab has no time, please contact his responsible postdoc.
- Teamleaders will write questions concerning the analysis of their projetcs in the dropbox file.
  - Deadline: Saturday! Prof.Hoffman has time before August 1th or after 27th.

Date for Interview with M94.5 (radiostation)
- must be a Sunday form 15.45 to 16.30 (Nadine) Date 19.07.2012 David&Nadine will join (perhabs one additional person).

Group T-shirts (Nadine)
- The next meeting will be used to decide which logo will be printed on our clothes.

Movie and politics (Jara)
- fixed Dates? Film will be produced on Saturday/Sunday 25/26th august.
- Tuesday 18th September 18h is fixed date for talk with politics. Possible locations: ZIEL II at Prof. Haller or H17. H17 is preferred.
- Fabian reports about contact to german consumer protection to Jara. - Possibly invite to interview?
- 3 politicians have confirmed their visit.
- Invite intern experts: Prof Pfaffl or Prof. Schnieke
  - Prof Skerra will be asked to decide which Prof he would recommend to invite.
  - Every team member should think about possible discussion topics.

Report School visit (Jara/Nadine) the school visit was very successful!

The printing of beer labels will cost 1€ per paper sheet in the copy shop.

Dennis will design some samples for team logos until next week's meeting. Let Prof. Skerra check these.

Tuesday July 31st

Present: Nadine, Saskia, Georg, Jara, Jeff, Dennis, Lara, Simon, Mary, Andrea, Martin, Alois, Fabian, Volker, Ingmar

Missing: Daniela (out of town), Roman (studying), David (defense of diplome on thursday), Katrin (studying)

25.8 - action day - what should we do for that?

(Fabian)

advanced as Nadine has to go earlier
do we want to organize this, Nadine + Jara could do this.
material is available from bmbf/biotechnologie.de
write press release
connect with other teams

Meeting with Prof. Hofmann

All teamleader should be present

Lab Report + New milestones

(all)

Coumaryl

Milestone from last meeting: Successful transformation of yeast.
- two transformations were successful
- meeting with Doreen for troubleshooting
- in the future: use positive control (GFP vector from Simon)
Did David contact Andreas Reichert for external sequencing?
- Ms. Lerchner could also know more.
New Milestone: Transformation of yeast for remaining enzymes
- Prof. Skerra suggest in vivo assays instead of purification assays, as purification is difficult and time consuming. Were are only interested in the in vivo activity!
- Western Blot to prove expression of enzymes. (should be possible for accuracy at least 5 AS with SDS-PAGE)

Limonene

Milestone from last meeting: Successful Ligation in pYes2 RFC 25 and pSB1C3 of both limonene synthase sequences.
- Quickchange was successful (Schwab)
- BioBrick version was more problematic (PCR product probably was faulty)
New Milestone:
- citrus: transformation of yeast (pYES2)
- lavendula: transformation of yeast until next Thursday (pYES2)

Caffeine

Milestone from last meeting: start of cloning in pYes2 and pSB1C3.
- Waiting for gene-synthesis
- If all enzymes shall be expressed from one vector, we could already start preparing this vector!
  - What is the maximal size of a yeast vector? Complete vector should be around 13 kbp.
  - Prof. Skerra suggests multiple transformation steps instead of one large vector.
    - Problem to get several restriction markers.
    - Mary will ask Doreen.
New Milestone: Finished Cloning Scheme. All three genes in one vector.

Thaumatin

We do not know how to get the antibody. (get this done fast!)
- Antibodies are extremely expensive -> half quantitative assay by gel scanning
  - New Milestone: Check for antibodies.

Light Switchable Promoter

3 ligations did not work.
Jeff wants a rfp generator RFC25 (Georg will do it)
Harvard BioBricks reordered.
New Milestone: Only ligation -> about 2-3 weeks if everything works + biobricks arrive

Constitutive promoters

Milestone from last meeting: Test the activity of TPI on ethanol dependency.
Characterization with limonene/thaumatin
Milestone: pYES2 should be finished

Prof. Skerra will be absent the next 3 weeks

Write short summary with substrate, amount, price if something is needed.

General Strategy to cover Methods

Genome Integration: Limonene
Purification: Thaumatin
Gas Chromatography: Limonene
Enzyme Assays: Coumaryl, Caffeine
Mass Spectrometry: Coumaryl, Limonene
Expression of multilpe genes from plasmid: Caffeine, Light Promoter
Brewed Beer: Limonene, Thaumatin
Regulation via external Stimuli: Light Promoter
Promoter Characterisation via ELISA: Thaumatin
Western Blot: use Strep Mab Classic Antibody --> ask Ina Theobald

Report Genome Integration

(Martin/Alois)

wanted to look for information concerning protocols to this and the second ordered BioBrick
- second BioBrick is no insertion vector
Does a commercial version for genome integration exist?
- Several do exist
- Principle is always the same
Which maximum length can inserts have for successful insertion?
- No upper bound
We only plan to integrate limonene
Is there something like the rfp generator for yeast?
Dennis will try iGEM integration vector
Martin will check whether everything is available for the protocol
Combine genome integration with knockout of enzyme which converts Geranyl-pyrophasphate. (Check work of Jay Keasling) (Martin and Dennis will check literature)

Brewing process of our Yeast (Martin)

When has David time for the brewing-process?
- We should start brewing in 2 weeks.

Report CAS-Conference

(Fabian)

Cooperation with Bielefeld/Frankfurt/Tuebingen (pYES2 Vector). Ingmar will take care.
Check toxicity of substrates/intermediates
Interview with London, only if they contact us again.
Check Erasynbio for sponsoring (Fabian will send mail to David)

Yeast Media and Trafo-detergenzien

Who can help?
- Alois will do it.
Ask Mary before preparing plates

Report of Interview with Prof. Schwab

(Lara)

Characterization is possible in their Lab
We need our own substrate

Interview with Prof. Hofmann:

Questions complete? Date (Nadine)?
All teamleaders should be present.

Panel Discussion

(Jara)

18.9 17:00 HS17
20 minutes presentation of our project
20 min talk by representative from the
20 min scientific talk by an expert - maybe invite somebody from the Netherlands.
˜ 1h panel discussion moderated by Prof. Skerra
- Student + Expert + Hofmann + 3 Politicians + Gierl
Beer + Brezn afterwards
Flyers?

Documentation

Matthias Mörch can help us with several things
- Chemical Structures
- Videos of us in the lab

Logo

(Dennis)

(postponed)

Tuesday August 7th

Present: David, Ingmar, Dennis, Daniela, Andrea, Lara, Jeff, Nadine, Mary, Fabian, Volker, Roman (late)

Missing: Jara (@ home, will join over skype), Martin (sick - if there are questions to be answered by me, I will take position to them afterwards in the protocol), Saskia (out of town), Alois (out of town), Katrin (out of town), Simon (only here for the first 30 minutes), Georg (exam)

Problems with Weihenstephan

(Jara over Skype)

Letter from Weihenstephan
- We must clarify that we are not associated with Weihenstephan.
- We must not use their slogans.

Panel Discussion

(Jara over Skype)

Room is reserved
Should we still serve beer & brezn or sparkling wine?

Substitution for Mary (August 12th - September 2nd) and Simon (August 20th - approx September 15th)

Somebody has to lead meetings
- Fabian can do it on August 21th
- Lara will do it on August 28th
Everybody should note absence in wiki

Lab Report + New milestones

(all)

Coumaryl

Milestone from last week: Successful transformation of all enzymes in yeast and try to express them.
- All transformations were successful
- All colonies did grow
- Western blot will be done tomorrow
Milestone for next week: 2 Quickchanges + further characterization (during next week)

Limonene

Milestone from last week:
- citrus: transformation of yeast (pYES2)
- lavendula: transformation of yeast until next Thursday (pYES2)
  - Quickchange was successful
  - 2 bad mini-preps
  - 2 parallel ligations of both enzymes in psB1C3 and pYES2 tonight
Milestone for next week: Transformation of yeast

Caffeine

Milestone from last meeting: clarify if enzymes in one vector or in three and an cloning scheme
Milestone for next week: Finishing cloning scheme

Light Switchable Promoter

Milestone from last week: Ligation
- Bad ligations from last week now worked
Milestone for next week: Enzymes for chromophores in pYES + work on fusions

Constitutive Promoters

Milestone: pYES should be finished

Thaumatin

Milestone: How will you do it with the antibody? did you order it?
- --> last proposal by Prof. Skerra was to analyse the supernatant first before thinking about ordering the (expensive) antibody. [Martin]

Talk with Doreen about Yeast

(Mary)

Our Yeast has auxotrophies for Histidin, Leucin, Tryptophan and Uracil
- Jeff has Tryp and Leu2 vectors
She did not know about maximal plasmid size

Yeast Media, Trafo-detergents and Yeast transformation - short explanation

(Mary)

Some modifications + annotations to the protocol for yeast transformation
- See annotated file in dropbox (pYES2_manual_Kommentiert.pdf)
- Please note that some preparations need to be done the day before, so plan carefully

Report Genome Integration

(Martin/Alois)

Conversion of geranyl-pyrophosaphate?
More informations for specific integration of limonene and thaumatin?
(postponed)

Brewing process of our Yeast

(Martin)

When has David time for the brewing-process?
- --> waiting for a detailed timetable. when will we have sufficient transgenic yeast to brew a limonene/thaumatin-beer? [Martin]

Lab Organisation

Who is helping when things are empty (Medien, competent cells, ....) ? - look in wiki!
Cleaning personal for lab material is on holidays, so maybe someone of our team can help with that!

Ordering substances (Mail to Prof. Skerra)

(Volker)

Volker will send the mail
David has a list of things for which did not find sponsors
- Some substances are only needed if enzymes were successfully expressed
Include Age1 in this list as stocks are almost exhausted
- Please only use Age1 if necessary!

Sequencing of biobricks - do we get sponsoring?

(David)

Talked to Ms. Lerchner?
No sponsoring
Ask other institutes wheter we can use their GATC accounts.

Yeast Transformation Kit from Invitrogen

(David)

(S.c. EasyComp Transformation Kit) - do we get sponsoring?
- No Contact possible.
- Doreen suggested the use of this kit.

Logo: Logo-suggestions

(Dennis)

No News
Ask other people.
Super Yeast with TUM Logo (Volker)
Lara can organise wooden chains with our logo

Interview with Prof. Hofmann

(Nadine)

questions complete? Date ?
- No contact possible

Action-Day on 25th of August

(Nadine)

Time limit 4 hours
Size limit 4,5 m^2
David will write a text for a flyer
We have 10 cards for the Munich zoo.
Doodle for 25th, 26th who has time
We will get a comic

Innovation Contest of Brau-und Lebensmitteltechnologie of TUM

Do we want to join? who wants to take the responsibility?
- Roster of teams is already full

Business Plan for our product

Ask Braufässchen for Data to work on

Tuesday August 14th

Present: David, Nadine, Katrin, Alois, Jeff, Ingmar, Simon, Martini, Fabi, Georg, Andrea, Volker,

Missing: Daniela (out of town), Lara (out of town), Roman (out of town), Jara (out of town, Simon will tell you about my news or see Dropbox), Saskia (out of town), Mary (vacation), Dennis (?)

Discussion: Unequal contribution of team members. How to handle this

Suggestion Mary & Simon: At least 30 whole days of labwork necessary per person (except Jara, Nadine, Volker, Fabi, David)
voting: 6 pro 5 contra 1 abstention (accepted after revoting)
- Every day every teamleader writes a list of things to do.
- Every morning at 9am things to do will be distributed among people
- If somebody arrives later ask the lableader (to be assigned) or teamleader
Main goal is that everybody is present everyday

Lab Trouble

Do not use the competent cells prepared by Roman
- Old ones will be thrown away. (Katrin will do)

Introduction: How to enter Biobricks in registry

(Simon, Volker)

- 2 People will do it, so item was dropped

Lab Report + New milestones

(all)

Ethanol-Promoter (Simon)

Report on Bachelor Thesis
- Still needs bricking (Simon/Ingmar)
  - Combine this with Limonene

Coumaryl

Milestone from last week: Western Blot, start quickchanges
- Results from Western Blot were bad
- Repeat at several timesteps (around 4h-12h) and change protocol
- Quickchanges were started
Milestone for next week: Improve Western Blot + Enzyme Assays + Finish Quickchanges

Limonene

Milestone from last week: Transformation in yeast
- Ligation did not work
Milestone for next week: Ligation + Transformation

Caffeine

Milestone from last week:
- Sequences have arrived, transformation of diluted sequences and cloning in pSB1C3 and pYES2 new is in progress.
- After having successfully inserted in pSB1C3, we will start to clone the genes together with promoters and terminators from Georg as soon as he has sequenced them.
- Cloning was started
Milestone for next week: Cloning

Light Switchable Promoter

Milestone from last week: Enzymes for chromophores in pYES + work on fusions
- Fusions worked, enzymes were transformed in bad cells
Milestone for next week: continue (2 more weeks)

Constitutive promoters

Milestone from last week
- Transformation was done with Romans bad cells
Milestone for next week: continue (cloning in pSB1C3/pYES2) + sequencing

Thaumatin

Milestone from last week: -
- Gene synthesis arrived
- Bad puffer was used so cloning has to restart
Milestone for next week: Transformation in Yeast completed

Logo Presentation

(Volker)

color: tum-blue
postponed

Gene Sequencing

(David)

Everybody is on summer holidays
Organize over Schwab Lehrstuhl.

Interview with Prof. Hofmann

questions complete? Date (Nadine)?
- Answer until Thursday

Report Genome Integration

(Martin/Alois)

conversion of geranyl-pyrophosphate?
more informations for specific integration of limonene and thaumatin?
Martin/Alois can start with the work next week.
Start experiment with mOrange cassette

Brewing process of our Yeast

(Martin)

Generate data for presentation
Should start this week.

Action-Day on 25th of August

(Nadine)

Progress Flyers (almost finished, in dropbox)
Progress press statement (finished, David will check)
Who has time ? sufficient people in Facebook event
Photos: contact Matthias Mörch (Jara)
Comics from Switzerland

Panel Discussion

(Jara)

Who will do presentation?
2 Reporters
Invite Matthias Mörch
Think about advertisement

Video from [http://www.wissenundkonzepte.de/ Wissen und Konzepte]

Okay
Invite to Panel Discussion

Establishment of glycerol- stocks?

Not necessary

Amsterdam

Shouldn't Doreen & co. come with us? At least we should invite her, because I think Doreen has really helped us until now and I have told them of Amsterdam and the US final before "recruiting" them for our team (Roman)
Postponed

Tuesday August 21th

Present: Volker, Jeff, Roman, Saskia, Alois, Jara, Daniela, Georg, Nadine, Fabi, Simon, Lara, Andrea, Dennis (later), Martin (later)

Missing: Mary (Vacation), David, Katrin (working), Ingmar (out of town)

Lab Report + New milestones

(all)

Coumaryl

Milestone from last week: Improve Western Blot + Enzyme assays + Finish Quickchanges
- Cloning: Quickchanges complete of: CHS, 4CL (1 missing), also sent to sequencing, all genes completely cloned in pYES2 and pSB1C3.
- Correct sequences available in silico --> Can be added to registry
- Characterization:
  - Expression of Enzymes: western blots were done, but ambiguous results. Find out optimum time for expression: Samples have been collected after 2, 5, 6, 8 hours, but western blot still has to be done. SDS-PAGE and western blot will be done by Daniela. Once we know a good time, we can make a 2 L batch so that we have enough enzyme for characterization. --> Make enough induction medium in 2 L flasks and autoclave it the day before! Also: Make 50 mL flasks with induction medium (autoclaved) for pre-cultures.
    - PAL: Tyr as substrate, details for enzyme assay still have to be determined. Detection of product @ 313 nm.
    - 4CL: Substrate have been ordered - ask Irmi if they have arrived. Add strep-tag --> purification and in vitro assay possible.
    - CHS: No way to buy/produce 4-Coumaryl-CoA --> only detection by western blot.
Milestone for next week: Quickchanges and sequencing done. Western blot for expression time profile done.

Limonene

Milestone from last week: Ligation + Transformation
- Still problems with cloning --> Colony-PCR for screening of clones
- 2 positive clones (one of each limonene synthase, citrus w/o consensus sequence, lavendula w/o N-End modification but with consensus sequence = 2 out of 5 possible genes) in pSB1C3 have been found --> Subcloning in pYES2 will be easier this way
  - For presentation @ Amsterdam: Present findings: different organisms, N-End rule, consensus sequnece --> Quantitative expression data would be great - Comparison of different genes with resulting enzyme activity.
- Waiting for sequencing results
- If you still have problems: Ask Roman about pGMT
Milestone for next week: Cloning complete & Transformation in yeast

Caffeine

Milestone from last week: Cloning
- Ligations done
- Transformation done
- Analytical digest showed that cloning was sucessful (All genes now in pSB1C3 and pYES2)
Milestone for next week:
- Sequencing, transformation in yeast
- Small scale expression and detection of enzymes by western blots (until August 28th).
- Also: Start 2 L batch (pre-culture needed!) and collect and freeze cell pellets to have enough enzymes for assays.
- Also: Start cloning of construct "Promoter 1 - gene 1 - terminator 1 - p2 - g2- t2 - p3 - g3 - t3" --> For expression of whole pathway in one yeast strain.

Light Switchable Promoter

Milestone from last week: continue (2 more weeks)
- 1 fusion construct (Gal-AD with Pif3) complete (already in yeast expression vector pGADT7), the other one is still being cloned
new milestone = old milestone ;-)

Constitutive Promoters

Milestone from last week: continue (cloning in pSB1C3/pYES2) + sequencing
- Promotors ADH1 und TEF1 in pSB1C3 (sequencing pending)
- Problems with TEF2 terminator
- Terminators: ADH1 and TEF1 in pSB1C3 (sequencing pending)
- Problems with cyc1 terminator
- Problems with deletion of Gal1 promotor from pYES2
Milestone for next week:
- Sequencing of finished constructs
- Solve problems (esp. w/ pYES2).
- Characterization via limonene and thaumatin desired by Skerra
- Maybe do characterization with GFP ("quick and dirty")

Thaumatin

Milestone from last week: Transformation in Yeast completed
- Problem: XbaI and SpeI chosen for cloning
- Status now: Cut vectors have been dephosphorylated (using SAP, fast-AP is better!)
- low yield in gel extraction
- ligation has been done
- trafo in E. coli complete,
- Next step: analytical digest & sequencing, transform in yeast (if analyt. gel ok), expression of Thaumatin
Milestone for next week:
- Expression of Thaumatin (first detection via SDS-Page of supernatant (and also of cell lysate),
- If positive: buy antibody - try to get it sponsored,
- BUT: shipping time! - talk to David, also detectable by flavour :)

EtOH-Promoter

Quickchange & PCR
- Talk w/ Ingmar
- Martin can help

Genome Integration

Milestone for next week:
- Integrate the mOrgange generator in yeast via BBa_K300001
  - cloning of a project relevant expression cassette in BBa_K300001 (thereby replacing mORgange genereator)

Upcoming Events

asap: Sign up for practice session!
- done: 21:30, lecture room HG-0G08
September 7:
- Regional Jamboree registration & attendance fee due
- Track selection due: Tracks chosen: 1.: Food/Energy, 2.: Health/Medicine, 3.: Foundational Advance
  - Send email to iGEM HQ
Project abstracts due: Project name ideas "TUM-Brew: iGEM's first and finest SynBio Beer"
Team rosters due - delete Jonas, add David (brewer) and Matthias Mörch (video, fotos, etc.)
Safety questions due - Simon will start with this

Publicity Page (2012.igem.org)

Jara will post news articles about us

Newspapers

(large ones): SZ, Spiegel, FAZ, Zeit, .... contact authors directly who have already written about iGEM.

Logo Presentation

(Volker)

Additional details / changes: Black shoes, grey beard, add iGEM gears on "Maßkrug" and/or "Lederhosn"
Add title / subtitle

Gene Sequencing

(David/Roman)

Sponsored by Eurofins
2,50 Eur per Read (800 - 900 bp)
30 free reads already used
New barcodes have been bought (inform Jeff if we are running out of barcodes)
Ask Roman and Jeff about details (where to find sequence results, how to prepare samples to send them off to sequencing)

Progress Wiki

(Fabian)

- each team must check and improve the texts on the official wiki concerning their projects - start early with this, it will have to be done anyway.
common structure:
- 1 paragraph as introduction
- well-sturcutred overview
- contents should be no longer than 3 or 4 pages (judges won't read overly long texts)
- include links to experimental data to prove statements.
Wiki design will be inspired by oettinger website
- Good photos of beer for background needed - buy it from a photo agency / stock photo page (20 or 30 Euros should be within our budget) or take them ourselves?

Interview with Prof. Hofmann

(Nadine)

questions complete? Date ?
- no response - still waiting for a contact person
- Ingmar and Volker will walk over there and take care of this

Progress: Genome Integration =

(Martin/Alois)

We have neomycin
buy SbfI from fermentas

Amsterdam

Trip is fully organised for those of us who stay from Thursday till Monday - What about the others?
- Roman, Nadine and Katrin will come later - either by night train (arrival in Amsterdam around 9 am) or by rental car (cheaper, more reliable, earlier arrival in Amsterdam - method of choice). Departure will be on Friday around 6 pm. Roman can drive over night (8 hours)
- Roman, Andrea will leave earlier on monday
Discussion about 30 days rule:
- General conflict between achievement of goals of team and personal interests.
- Skerra will make final decision who will go to Amsterdam and who won't.
Should'nt Doreen & co. come with us? At least we should invite her, because i think Doreen has really helped us until now and i have told them of Amsterdam and the US final before "recruiting" them for our team (Roman)
- Do we have enough money to invite them?
- On the one hand they always answered questions if we asked them, but they did not contribute directly or without being asked first
- Many members of Skerra group contributed more than they did.
- Idea: Offer them to pay attendance fee but ask them to make sure that the Schwab-LS will pay their travel expenses

Brewing process of our Yeast

(Martin)

Latest news from David Herrman: He never wrote explicitly if he will do the brewing for us. Now he claims that he never offered to brew for us - at this point we don't have a way to brew.
Possible solutions:
- "brew" in a 2 L flask or in a fermenter - no real beer
- Ask "Braufässchen" if they can help us out - Mary has personal contact to them but is on vacation until September 2nd or 3rd - who will contact them? - Jara
- Ask "AWW"

Report Funding

(David)

- 625 Euro per person have been granted for Amsterdam from "Studiengebühren" from WZW - we will probably get more for Boston

Progress Report Action-Day on 25th of August

(Nadine)

Preparations finished (except "Stellwände") - will be organized by LMU
we will present our CAS-poster, LMU team has a poster, also: pictures for children's "Malwettbewerb"
Lara, Andrea, Dani, Martin, Saskia, Jara will attend
4 ppl from LMU
information material will be printed at Skerra LS
time: 8:45 a.m.
Matze will be there and take care of documentation (fotos, maybe video)

Progress Report Discussion Round

(Jara)

Presentation for our project etc. will be done by Jara and possibly one additional person (Ingmar? Martim? Lara? Mary? Volker?)
Who will answer question regarding our project during discussion round (questions will be about our project, general questions concerning GMOs & food will be answered by our experts (Prof. Wenzel,...))?
Maybe invite one person who is well informed and has scientific knowledge but is against GMOs - who? "Gentechnik-Bundesbeauftragter der Grünen" - Volker, "Gentecchnikfreier Landkreis", "Greenpeace", "B.U.N.D."
Financing for travel expenses of person of BVL - our budget should include 1000 Euro for PR expenses - check with David

Progress Report Video

(Nadine)

Video will be made by "WissenUndKonzepte" in German - They made a script for this
2 Team members needed as "actors" - who?
6 or 7 additional ppl as "extras" during labwork
Wash "Laborkittel"!
Matze can make an additional video in English - maybe a 1 minute trailer - good for presentation

Comic

(Nadine)

Almost done, some texts still missing
In dropbox :-)
General presentation of iGEM and short project descriptions

Tuesday August 28th

Present: Volker, Lara, Jara, Saskia, Daniela, Andrea, Roman, Jeff, Dennis, Ingmar, Georg

Missing: Mary (Vacation), David, Nadine (vacation), Martin (Bomb near his home), Fabian(Vacation), Simon (Vacation)

Upcoming Events

September 7:
- Regional Jamboree registration & attendance fee due--> Will be decided next week in hope it is clear which teammembers will travel to Amsterdam and which won't. Volker will care about this.
- Project abstracts due
- Track selections due -> email sent?

Lab Report + New milestones

(all)

Coumaryl

Milestone from last week: Improve Western Blot + Enzyme Assays + Finish Quickchanges
- Quickchanges are finished and have been check by sequencing.
- But sequencing showed additional new mutations so that new quickchanges have to be done.
- Western Blot has been repeated but no desired effects were detectable.
  - Possilbly repeat the experiment with a PVDF membrane.
  - Do a gene expression in larger scales in order to purify the cell extract with magnetic beads.
  - Test emulgatores and a pH~7,5-8 to increase the solubility of tyrosine.

Limonene

Milestone from last week: Ligation + Transformation
- Milestones were completely reached!
New milestone:-

Caffeine

Milestone from last week: Cloning
- Cloning is completed!
New milestone:
- transformation in yeast
- start with first expression experiments and begin promoter fusions

=Light Switchable Promoter

Milestone from last week: continue (2 more weeks)
- Milestone is reached.
- Work will continue with transformation in yeast.

=Constitutive Promoters

Milestone from last week: continue (cloning in pSB1C3/pYES2) + sequencing
- Tef1, Tef2 and ADH are ready to use and already sequenced except ADH.
New milestone: begin with characterization of the promoters using thaumatin and GFP.

Thaumatin

Milestone from last week: Transformation in Yeast completed
- Transformation failed two times and will be repeated.

EtOH-Promoter=

Quickchange & PCR

Check your passports!

Logo

Slogan/title
"TUM-Brew: iGEM's first and finest SynBio Beer"?
- Both will be decided finally next week in presence of Prof Skerra.
"Trachten"/Shirts/Gingerbread?
- David may contact the TUM sponsoring shop to check the opportunities of free shirts/jackets. Beside this we will keep our plan to present ourselves in trachten and gingerbreads.

Interview with Prof. Hofmann

Ingmar will inform Prof. Skerra about what analytical devices we need. Prof. Skerra will line up all necessary measures and contact Prof. Hofmann.

Progress: Genome integration and brewing

(Martin)

Is genome integration necessary for brewing?
- Yes, it is.
- As mOrange is already included in the genome integration vector, we will try to integrate it stable in yeast.

Report Action-Day on 25th of August

(Jara)

Drawing competition winners?
- We will contact all of our ten winners. Moreover the action day was a success as many interested people have been informed about biotechnology.

Progress Discussion Round

(Jara)

Who will answer the questions?
- As Jara found no drink sponsors we will likely buy them ourselves.

Progress Video

(Nadine, Jara)

- The screenplay is in work. It was proposed to produce the video after wiki freeze.

Comic

(Nadine)

@@ Line 194: / Line 194: @@
 </div>
-==Wednesday, April 4th==
+==Wednesday April 4th==
 <div id="MWednesday_April_4th meeting">
 ==='''Overview'''===

Team:TU Munich/Notebook/Meetings

From 2012.igem.org

Revision as of 13:33, 31 August 2012

Contents

Meetings

Tuesday March 6th

Presentation of Research Results in our internal Wiki

General Remarks

Promoters

Ethanol-inducible Promoters

Inducible Yeast Promoters

Biosynthesis of small molecules

Resveratrol

Xanthohumol

Raspberry Ketone

Caffeine

Aspirin

Nicotine

Strawberry flavor

Colors and pigments

Beneficial Peptides and Proteins

Knottins

Thaumatin

Lactoglobulin

Discussion & Next steps

Decision on structure of project

Next Steps

I) Detailed literature research

II) "Taskforce Vector Design"

Interviews

Miscellaneous

Tuesday March 20th

Overview

Interviews

1. Dr. Zankow, Beverage Oriented Biotechnology

2. Prof. Schwab, Biotechnology of Natural Products

3. Prof. Hoffmann

Fundraising

Human Practice

TO DO

Wednesday April 4th

Overview

Human Resources / General topics

Projects

Light-inducible Promoter (Jeffrey)

Constitutive Promoters (Georg)

Ethanol-inducible Promoter (Simon)

Coumaryl-CoA (Ingmar)

Next steps

Monday, April 16th

Organisation

Round of Introductions

Press

Logo

Comments by Prof. Skerra

Research

Prof. Schwab

Coumaryl

Limonene

Caffeine

Thaumatin

Gene Synthesis

Promoters

Saturday

Saturday, April 21th

Introduction by Volker into BioBicks and Methods

Team forming

Next Meeting

Tuesday May 1st

General

To Do List

Sub- Team Reports

Human Practice / Press / Video

Ethanol inducible promoter

Light-inducible [romoter

Limonene

Caffeine

Thaumatin

Next Meeting

Tuesday May 15th