Team:Wageningen UR/Notebook

From 2013.igem.org

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Revision as of 13:46, 2 September 2013

notebook entries

Week 16

September (16.09 - 22.09)

Science Cafe Wageningen

16.09.2013

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Week 15

September (09.09 - 15.09)

Week 14

September (02.09 - 08.09)

Week 13

August (26.08 - 1.09)

iGEM Netherlands

26.08.2013

Performed some more Gibson assemblies and tried to ligate DH and MT together in the same vector. Made some more chemically competent cells and the primers for our mini-genes arrived.

Week 12

August (19.08 - 25.08)

iGEM Netherlands

24.08.2013

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Gibson assembly

22.08.2013

The colony PCR’s of DH, MT, ACP and eforRed are successful. Performed another Gibson assembly with KS.

Gibson assembly

21.08.2013

Today we did our first Gibson assemblies of DH, MT, ACP, eforRed and ATP. Including transformations.

Working on the Wiki

20.08.2013

The wiki is making good progress, everybody should be able to work with the system now. Objectives are to upload content, look for icons that can be used in the menu bar and to finish the team page

Transformations

19.08.2013

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G-blocks from IDT arrived today

19.08.2013

Today the G-blocks we ordered finally arrived! This is great news, because now we can start on the gibson assembly and transformations.

Lovastatin Pathway

19.08.2013

The lovastatin pathway has been added to the metabolic model of Aspergillus niger. Because the medium composition of the model is not yet properly defined, the maximum flux towards lovastatin can be achieved. This however is not realistic and therefore the next thing is to redefined the medium composition.

- B.D. Ames et al., 2011. Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis. PNAS, doi/10.1073/pnas.1113029109

Week 11

August (12.08 - 18.08)

iGem Olympics: Second round

18.08.2013

We played pool during the second round of the iGem Olympics. Also to celebrate Shreyans Birthday!

Molecular Interactions Symposium Berlin

14.08.2013

We went to the Molecular Interactions symposium in Berlin to present a poster about our project! We were kind of surprised that there were no other iGem teams. The interaction we had with the interested people lead to a couple of nice suggestions to the poster and the project. We even had a good discussion about Gibson Assembly. The interesting speakers there, nice ambience and good dinner in the evenings these 2 days totally made up for the 2 times 7 hours of midnight driving!

Title

00.00.2013

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Title

00.00.2013

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Week 10

August (05.08 - 11.08)

Vitruvian man stamp

11.08.2013

We looked into using Vitruvian man metal stamps to make imprints in the agar.

Workshop by Paulien Poelarends

09.08.2013

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Protoplasts

12.08.2013

During this week we have prepared media and spores to make protoplasts. The protoplasts will be used for transformations. Until recently Novozym has been used to make protoplasts, this is not available anymore and some new protocols have to be used.

Lovastatin

06.08.2013

The restriction enzymes that we ordered last week have arrived. After a few attempts the sequences have been optimized and the G-blocks can be ordered. Also some Lovastatin has been ordered to perform some experiments with A. niger. Lovastatin may be fatal to cell growth of A. niger therefore experiments have to indicate whether A. niger is resistant to Lovastatin. Otherwise A. niger has to be transformed with the resistance gene from A. terreus.

Expanding the database

05.08.2013

In order to expand the database, more information on secondary metabolite backbone enzymes from Aspergilli needs to be added. Luckily there is a recent publication on secondary metabolites from A. nidulans, A. fumigatus, A. niger and A. oryzae.

- D.O. Inglis et al., 2013. Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiology, Vol. 13, p. 1-23.

Week 9

July (29.07 - 04.08)

iGem BBQ

02.08.2013

We finally had our first group BBQ! Together with all people that are involved with our team! It was a really nice day.

Metabolic modeling

30.07.2013

The current metabolic model of A. niger needs to be expanded to include the lovastatin pathway as known from literature. First the model has to be checked: is it balanced? can we perform a FBA on the current model?

- M.R. Anderson et al., 2008. Metabolic model integration of the bibliome, genome, metabolome and reactome of Aspergillus niger. Molecular Systems Biology, Vol. 4, Article number 178; doi:10.1038/msb.2008.12

Shopping

30.07.2013

This week we ordered the restriction enzymes for the assembly strategy. Also other stuff should be ordered like pJET, lovastatin, A. niger & A. terrus strain, Gibson assembly kit...

Week 8

July (22.07 - 28.07)

Obtained E.coli expressing chromoproteins from Braunschweig

25.07.2013

We obtained the E.coli from Braunschweig that have plasmids with an chromoprotein encoding gene insert. Lets start introducing it into Aspergillus Niger!

Metabolic activity: GFP

24.07.2013

In a similar way that the DAPI stained cells give an indication of metabolic activity by showing an increase in nuclei, GFP-transformed A. niger also give an indication of metabolic activity by giving an indication on whether transcription and translation occur.

Splitting up the domains

22.07.2013

Luckily, we got some help from Kal, a really nice guy who offered a lot of help. Several very useful websites he suggested could analyse the AA sequence and give some advice on where is the possible site to split the whole protein. We combined the analysis result with references, we finally got a clear idea (or at least we believe) about the boundary of the domains.

Week 7

July (15.07 - 21.07)

Working on the Wiki

20.07.2013

The design of the wiki is making good progress. Basic layout has been determined and we created icon to indicate different parts of the project.

Article searching

18.07.2013

Totally lost in how to divide each single domain in the lovB gene. By searching the articles, we could find information and experimental details about ACP, KS, MAT and CON domain. While for the others, still virgin land. KR seems to have two subdomains, what the hell~

Database design

16.07.2013

text here

Week 6

July (08.07 - 14.07)

Lovastatin strategy

12.07.2013

Now that we have the general idea the details need to be worked out. Therefore we came up with a strategy to assemble the modules using compatible restriction enzymes. The strategy will enable us to assemble several modules and to have a stop codon at the end of the assembled gene without a frame shift. Also the general planning of the project was worked out this week.

DAPI staining

11.07.2013

It has been found that dormant conidia are predominantly bi-nucleate (85%), the remainder being uni-nucleate. Therefore, if one stains the giant cells with DAPI, which colours the nuclei, one can assess whether the nuclei within the cells are actively dividing. This appears to be the the case and thus indicates that the cells are not in a vegetative state.

Week 5

July (01.07 - 07.07)

Science Café

02.07.2013

We met with the Science Café organisation to discuss the possibilities towards our team organising an evening there with a Biohacker theme.

BioKe meeting in Wageningen

05.07.2013

A BioKe spokesperson came over to Wageningen. We gave her a tour and presentation. We obtained the Q5 PCR kit!

Lovastatin

05.07.2013

Finally the search has begun, this week we have started with literature research for the Lovastatin project. Some topics were for example what is Lovastatin, which genes are involved in Lovastatin production in Aspergillus terreus, and how to measure Lovastatin production. The first idea was to introduce the Lovastatin pathway of A. terreus into A.niger. During the research we found that one of the bigger genes involved is composed of several domains. Our new idea now is to synthesize these domains, making several modules that we can assemble as we wish.

Database planning

01.07.2013

text here

- J.F. Sanchez et al., 2012. Advances in Aspergillus secondary metabolite research in the post-genomic era. Nat. Prod. Rep. Vol. 29, p. 351-371

Primers for septa visualizition arrived

2.07.2013

After some waiting time the primers needed for the visualization of the septa using a proton-ATPase finally arrived and this part of the project could also get started by isolating the gene from genomic A. niger DNA.

Sequensing results arrived

10.07.2013

The sequencing results from the actin gene arrived and everything looked fine. We had the right gene without mistakes and could go on with the actin cytoskeleton project.

Actin gene in house internal brick system

24.07.2013

The actin gene for cytoskeleton visualization purposes was ligated into a house internal brick system with a n-terminal GFP fusion. This construct was then introduced into E. coli for amplification purposes.

Week 4

June (24.06 - 30.06)

Calcofluor staining

24.08.2013

text here

Week 3

June (17.06 - 23.06)

Do It Yourself Biohacker Meeting 2

20.06.2013

We had a DIY Biohacker Meeting in the Waag, Amsterdam. The Waag had its grand opening and there was a exposition and party afterwards

Do It Yourself Biohacker Meeting 1

18.06.2013

We had a DIY Biohacker Meeting in the Waag, Amsterdam. Learned how to make your own potato-agar

Finding the right conditions for distinct phenotypes

22.06.2013

As described in literature, N593 formed giant cells after 24h at 44C. Since transcriptome data on N400 from multiple stages in its life cycle is available, this strain is chosen such that this research complements the current RNA landscape profile of A. niger and allows for comparison of data from different life stages. However, unlike N400, N593 repeatedly formed mycelium at 44C. To overcome this effect the temperature was increased to 45C, at which a single cell phenotype was obtained for N593.

Week 2

June (10.06 - 16.06)

Funny group picture

15.06.2013

Opportunity was presented to take a few fun group pictures... we obviously took it

Dinner and discussion

14.06.2013

We met at Mark’s place for dinner and evaluation of possibilities in using secondary metabolites for our project

Last and 8th recruit

13.06.2013

Yeng Ding AKA Danny joind our team. We are now 8 members strong!

ThinkerCell & CloneManager meeting

12.06.2013

The ones from our team who had learned to work with these software tools, during one of the iGem Netherlands courses, taught the others how use it.

Lab supplies

11.06.2013

We ordered reagents for cloning and PCR.

Collaboration

11.06.2013

We contacted the Cornell University iGem 2013 team to talk about possibilities for collaboration. They also work with fungus.

Host Engineering: Research plan

13.06.2013

Different environmental conditions induce changes in phenotypic cellularity of Aspergillus niger. Mapping reads onto the reference genome allows for discovery of patterns in gene expression that are unique to the single cell phenotype.

Week 1

June (03.06 - 09.06)

Actin primers arrived

26.06.2013

Finally the improved pair of actin primers arrived and after not getting good PCR results for some time I could finally get started and optimize PCR conditions for gene amplification of the actin gene.

House internal brick system

25.06.2013

The house internal brick system with a n-terminal GFP fusion was proven to be ok by restriction digestion and was ready to be worked with.

Host Engineering: Research Question

04.06.2013

Research question:
Finding sets of candidate genes causative to the single cell phenotype in Aspergillus niger by transcriptome analysis.

Subquestions:
- is A. niger metabolically active at 44C?
- does A. niger divide at 44C?

May

Meeting room

26.05.2013

We reserved a room for all future Monday and Thursday lunch meetings in the Forum

Visualization of the actin cytoskeleton

27.05.2013

Finally the labwork for the visualization of the septa and actin cytoskeleton started. And the first thing to do was order primers and isolate genomic DNA from A. niger.

Meeting iGem Uppsala

18.05.2013

We met with the iGem Uppsala 2013 team in Uppsala

Created a Gmail account

23.05.2013

We created a Gmail account. Let the spam begin

Created a Twitter account

15.05.2013

We created a Twitter account. Hello world! Meet iGem Wageningen 2013

Chromoprotein sequencing

13.05.2013

Looked into sequencing options: codon optimization to Aspergillus Niger, suffix/prefix, (illegal) restrictionsites. Let’s order some g-blocks!

Lab space

08.05.2013

We arranged our very own lab space!

Host Engineering: why?

20.08.2013

The single-cell phenotype Aspergillus has a higher surface to volume ratio and results in a lower viscosity of the liquid broth, offering perspective on substantially increasing process yields when used in liquid fermentations. Besides from an industrial point of view it is also interesting from an evolutionary perspective, which couples application-oriented research using a directed evolution approach to a more fundamental research topic: the evolution of multicellularity.

April

Good Lab Practice

24.4.2013

We had a lecture on good lab practice and safety in the lab

Task division

18.04.2013


Co-ordinator – Michiel
Secretary – Emiel
Sponsoring - Emiel
Safety - Jingjing
Lab manager – Shreyans
Strategy - Marit
Wiki – Michiel
Human Practice - Marjan

Influence of temperature on germination

20.08.2013

A paper from 1970 shows that at 44C Aspergillus niger does not germinate, but rather forms giant cells. This dimorphism is not unfamiliar within the fungal world, where at lower temperatures the mycelium is formed and at higher temperatures the yeast-like form is found.

Anderson, J. G. and J. E. Smith (1972). "Effects of Elevated-Temperatures on Spore Swelling and Germination in Aspergillus Niger." Canadian Journal of Microbiology 18(3): 289-297.

March

February

January

Title

00.00.2013

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

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