Difference between revisions of "Team:DTU-Denmark/Notebook"

Team Overview

The DTU BioBuilders had the first official meeting. Fun team building activities were planned and the members got the first real taste of what iGEM is really all about.

Team Overview

The annual BioBrick Tutorial was held and 89 exited and talented iGEM participants were gathered at DTU for an exiting weekend.

Team Overview

The team participated in Science in Forum, where they had a stand and told interested high school students about the wonders that is iGEM. The team based their explanations on the previous team's project and on the many project ideas that they had discussed during their time in iGEM.

Team Overview

Lo and behold! After many hours of discussing various projects and weighing the pros and cons of each of them, the DTU BioBuilders have now finally come to an agreement. This year the team will work on making a general tool box for uses of fungi. This entails ways of creating mycotextures to be used in the colonization of Mars.

Team Overview

The team attended the Nordic iGEM Conference hosted by Team Lund. Here they presented their project and showed off the amazing poster specially made for this event.

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Needless to say that everyone had fun seeing and discussing the other groups' projects.

Synthlab Overview

The first day in the lab was used for creating media of different types and concentrations for the species to be grown on for two reasons:

1. To illustrate an examine the growth rate (for modelling)

2. To multiply our pure species for use in liquid media

We used the media created in the "PDB (or PDA) and MEA media for species" protocol. It was also the plan to create Vogel's medium as well as a mineral salt medium as certain species had shown in the previous study to grow well. However, we did not have the proper materials to create those two and discarded the idea. We also came to this conclusion as we were recommended not to spend time on specific media just yet.

SynthLab Overview

20/6/2018
Pleuratus ostreatus was re-inoculated on the solid agar plates by placement of around 8 mycelia "balls" under a fume hood and left at 30 degrees Celsius. As to save time, fewer plates were inoculated and a YBD plate was included.

Today, we had gotten another species, Schizophyllum commune, in two forms: a wildtype as well as a mutant (Δsc3) which were also inoculated on PDB, MEA and YBD plates.

22/6/2018
The first sign of growth in our species was showing and pictures were taken of every plate as to count pixels and indicate the growth rate. Pictures were taken every morning for 5 days.

SynthLab Overview

26/6/2018
Aspergillus oryzae was received and inoculated on MEA, PDB and YBD plates (5 days of photographs will follow this species's growth in the same manner as the other species).
Today we decided that our species had grown enough and the samples were then removed and stored at a cooler temperature excluding those used for later liquid media.
Liquid PDB (PDA) was prepared for growth in our "brick"-molds (see further down) using the "PDB and MEA media for species" protocol without the addition of agar. 500 mL was created for each species. The PDB concentration chosen was the middle concentration in the table as these showed the strongest growth. At the end of this process, we should be ready to start with the actual growth of our building material.

Mycolab Overview

2/7/2018
Boxes: Initialisation of tests to determine best substrates and conditions for formation of a brick made of fungi. Saw dust was autoclaved.
Spore suspension: Spore suspensions are made for easy storage of spores. The spores are used for protoplastation and inoculation of boxes and plates. A. oryzae did not sporulate. It was incubated from June 26th to June 29th. Three days is not enough time for it to sporulate. P. ostreatus and S. commune did not sporulate either.
Reinoculation: Fungi strains were reinoculated onto new plates that all contained the same medium. Plates were checked for best growing conditions for the different fungi. PDA was in general the most promising medium and this was chosen as the medium used going forward. Three plates were inoculated for each strain with three-point inoculation. Depending on strain, different light and temperature conditions were used in incubation. The conditions were chosen based on knowledge of growth from research:

• S. commune (wildtype and ΔSC3): Two plates for 30°C light and one for 27°C light.

• P. ostreatus: Two plates for 28°C dark and for for 25°C light.

• A. oryzae: Two plates for 28°C dark and one for 30°C dark.

3/7/2018
Boxes: A. oryzae and S. commune were used for inoculation of boxes. PDA and mycelium with attached agar mixed. Inoculated medium mixed well with saw dust in boxes and incubated at 30°C dark for a week.

5/7/2018
Boxes: Pipette boxes inoculated with fungus/hay mixture from Skyttegaarden. 4 boxes inoculated, two with parafilm and 2 with the lid. Inoculation of boxes with species of our own.

6/7/2018
Photos of fungi: Photos were taken of the fungi every day to assess the growth. All species show growth although not all plates did. A. oryzae showed signs of sporulation.
Boxes: S. commune show growth regardless of medium concentration and volume. However, higher concentration of medium means more growth. A. oryzae boxes moved to 28°C dark. S. commune all moved to 30°C light as they seemed to thrive there.

Mycolab Overview

9/7/2018
All through week pictures of plates were taken everyday to follow growth and sporulation.
Spore suspension: Spore suspension made from plates from last week following the spore suspension protocol. Spore suspension ended up with a concentration of 1.69 * 10⁸ spores/ml.

13/7/2018
Boxes: New substrates used: white and brown rice and coffee grounds. Rice are natural substrates for A. oryzae and coffee grounds are supposed to add to the texture of the mycelia. All substrates autoclaved before inoculation of boxes. Pipette boxes inoculated and incubated at 30°C dark.
Baking of boxes: The baking or autoclavation of the boxes were to find a way to kill the mycelia, so the boxes could be brought outsite the laboratory. Boxes inoculated from Skyttegaarden and saw dust boxes were autoclaved. The saw dust boxes were very crumbly regardless of concentration before autoclavation.
Weighing of boxes: Boxes were weighed everyday to follow the growth of the mycelia and to see if there was a difference over time.

Synthlab Overview

20/7/2018
Experiment: First insertion of genes into a backbone
The purpose of this experiment is twofold. Firstly, to gain experience in using the basic techniques associated with molecular biology. Secondly, the scientific focus of the experiment is to perform an insertion of the MelA gene () and the CaMV 35S promoter (BBa_K1825004) into seperate backbone for amplification in E. coli This is followed by a miniprep to extract the plasmids. The DNA delivered by IDT was suspended in EB buffer (see protocol) Using part 1 and 2 from the BioBrick tutorial (Later adapted together with protocol 2) the parts were digested and ligated with the backbone from RFP (BBa_J04450)

Mycolab Overview

16/7/2018
Reinoculation of plates: A. oryzae inoculated on new plates in three point inoculation with spores from spore suspension. So far, inoculation has been done with mycelia from earlier plates. Three plates were incubated as normally in 28°C and one at 4°C to see how the fungus fares at lower temperatures.
Boxes: Rice boxes were quite hard after only two days in the incubator, which was viewed as a very good sign.

17/7/2018
Baking of boxes: After autoclavation the boxes were quite moist and therefore fell apart quite easily. In stead of autoclaving, microwaving or baking normally was suggested to avoid the bricks being too wet. Box from Skyttegaarden was put in the microwave. After 10 min it was still wet.
Protoplastation and transformation: Protoplastation was initialised according to the Protoplastation and Tranformation protocol

19/7/2018
Protoplastation and transformation: Protoplastation was initialised according to the Protoplastation and Tranformation protocol

20/7/2018
Boxes: Regardless of mycelia and media volume the brown rice inoculated with A. oryzae were crumbly and the mycelia had only grown on the stop of the brick. The white rice boxes (also with A. oryzae) were different according to volume of liquid media added to the rice. The less media, the sturdier the brick was to pressure from hands. Adding glucose to S. commune boxes with saw dust did not make a difference. The brick is still very crumbly.
Baking of boxes: Rice boxes baked at 90°C and checked every 15 minutes. After the fire 15 minutes the paper surrounding the brick was very wet. After discussion a new plan was formed: Rice is cooked when in water and surroundings are very hot, so we try to cook them quickly at high temperatures. Therefore: One rice box is baked at 90°C for two hours and then 200°C for one hour. After that the brick is baked 70°C O/N. The other rice brick is only baked at 70°C O/N. Saturday they will be taken out and photographed. See designated photo entry for photos of the bricks.
Small bricks: We have received ice cube trays to make smaller bricks, giving us the opportunity to make more bricks quicker and even different bricks at the same time. First small bricks are made with spores and different ratios of saw dust, white rice and coffee grounds to see how they work together. To avoid contamination every other well was inoculated instead of each well. See picture.

Synthlab Overview

23/7/2018
Experiment: First insertion of genes into a backbone
Competent cells were transformed using a transformation protocol. After the transformation, the cells were plated on LB amp plates (which in retrospect is the wrong kind of plates) and left overnight. The plates from Monday 23rd were not successful, as we used amp plates instead of cam plates. The protocol has been rewritten accordingly.
The leftover cells were plated on some new LB cam plates.

25/7/2018
Experiment: Preparation of 2nd round of genes
The purpose of this experiment is to insert the DNA delivered from IDT into plasmids for progapagation and further use. The genes that are to be inserted are; pGpdA, pTrpC, pTrpC-GFP and ScGPa. This will be accomplished as per the Adapted assembly protocol.

26/7/2018
New Gene have arrived: CaMV 35S Terminator, TS1 holology region, Hygromycin B. These were digested following the adapted protocol.
The ligation results was transformed and plated on LB cam plates O/N.

27/7/2018
Experiment: First insertion of genes into a backbone
The prepared O/N cultures were miniprepped (see miniprep protocol) in order to have the purified plasmids.
Experiment: Preparation of 2nd round of genes
The O/N plates produced 3 transformants and a correct positive control, but the 4 older digestions produced no colonies, which suggests that digestions was unsuccesful. Consequently, the digestions, ligation and transformation was redone. The transformation used 35 µL competent cells instead of the 50 µL the protocol requires. The liquid media was scaled down acordingly.

28/7/2018
Experiment: Preparation of 2nd round of genes
The plates containing transformants was moved to the fridge.

Mycolab Overview

23/7/2018
Boxes: New boxes made with different combinations of rice, saw dust and coffee. Tests with different kinds of treated rice initialised: porridge rice cooked, raw or grounded were autoclaved and so were jasime rice, raw (but cleaned), normally cooked, overcooked and grounded.

24/7/2018
Another spore suspension of A. oryzae made according to the spore suspension protocol.

25/7/2018
Small bricks: More small bricks made. This time from differently treated rice: porridge rice, normally cooked (1), jasmine rice, overcooked (2) and jasmine rice, normally cooked (3).
Rice mixed with spore inoculated medium and put into the wells in following pattern:

Incubated at 28°C dark.

26/7/2018
Small bricks: Same procedure for inoculating medium as 25/7. Other rice used:

1. Ground jasmine rice

2. Porridge rice

3. Ground porridge rice

4. Washing jasmine rice

5. Finely ground rice

6. 10 ml rice

Added to two trays. First 5 ml inoculated rice was added, in the second inoculated rice was added until full.

27/7/2018
Test of contaminations found in boxes: Contaminated saw dust and PDA plated to ID them. The contaminated PDA might be A. oryzae and the contaminated saw dust might be S. commune. This is interesting because we might have found a way to get spores from S. commune.

Synthlab Overview

30/7/2018
Experiment: Preparation of 2nd round of genes
O/N cultures have been prepaired. 2 for each plate
Experiment: Extraction of RFP from the kit
The purpose of this experiment is to transform the RFP gene (BBa_J04450) from the distribution kit into an DH-5-α compent E.coli and use a miniprep to extract it again.
Using the transformation (subpart 3) from the Adapted assembly protocol, four transformations were made; 1 RFP, 1 negative and 2 positive with (2 µL) A1. The transformants were plated on LB cam plates and cultivated overnight.

31/7/2018
Experiment: Preparation of 2nd round of genes
Overnight cultures were Miniprepped. Experiments were performed by Joen, David and Jacob.
This concludes the preparation of 2nd round of genes experiment.

1/8/2018
Experiment: Extraction of RFP from the kit
Two colonies from the RFP transformant plate were picked and placed in a liquid O/N culture.

2/8/2018
Experiment: Extraction of RFP from the kit
Overnight culture were miniprepped to extract the plasmids.
This concludes the Extraction of RFP from the kit experiment.
Experiment: Gibson assembly of basic toolkit
The purpose of this experiment is to assemble what we can of out basic toolkit using Gibson assembly. The parts to be assembled are; TS1, pTrpC, Hph, CaMV 35S terminator and TS2. The Gibson assembly will be performed according to the Hifi assembly protocol. As the second homology region, TS2, had not arrived at this point, we began assembling the other genes into a composite. Part 1 of the protocol was done (PCR amplification). Experiments performed by David, Joen and Jacob.

Mycolab Overview

30/7/2018
Reinoculation of plates: Cold A. oryzae grown to spores. Probably because of the cold incubator being broken. Temperature was around 20°C instead of 4°C.
Baking of boxes: Ice cube trays from 20/07/2018 photographed and baked. Baked at 70°C for 4 hours and 40 minutes and then photographed. Put bad to bake O/N.
Before baking:

After 4 hours and 40 minutes:

31/7/2018
Baking of boxes: Ice tray taken out after being baked at 100°C for roughly one hour.
Small bricks: New ice tray inoculated with spores suspension. Same method as last time. Incubated in fridge at 4°C.

2/8/2018
Death test: After baking we need to make sure the mycelia in the bricks are actually dead. That's what we're doing here. For the large bricks: Tape is touched onto brick to catch potentially alive mycelia or spores and touched onto plate afterwards. For the small bricks: Blue inoculation needles are touched onto the brick and inoculated on plates according to marks from 20/07.
Test of contamination found in boxes: Turned out to be nothing of interest.

3/8/2018
Baking of boxes: As the large boxes from 20/07 had been unprotected while they were in the office (after their baking) spores from the office may have landed on them and started growing. They are baked again at 100°C for two and a half hours.
Death test: Another death test in which the small bricks are broken in half and the inside is touched slightly on a PDA plate to see if the inside is dead as well.

Synthlab Overview

6/8/2018
Experiment: Gibson assembly of basic toolkit
Adding the homology regions to pTrpC, CaMV Terminator and Melanin for assembly into the basic plasmid after it's contruction. This is done via PCR amplification with primers containing and extra 20 bp upstream from the binding part of the primer.

7/8/2018
Experiment: Gibson assembly of basic toolkit
PCR products was gel-purified and nano-dropped

[note: the difference between "TS2" and "TS2 nedre" is that the gel had two bands for TS2] The Gibson mix arrived and we followed the Gibson protocol to assemble the basic plasmid. We assembled two different plasmids. One with TS2u and one with TS2n. The difference is from the gel electroforesis. One band was slightly further down than the other.We did not know which was the right one, so we collected both.
Experiments performed by Tenna, David and Jacob

8/8/2018
Experiment: Extraction of amilCP from distribution kit
A liquid O/N culture were prepared from the plated amilCP transformants. Addionally, replating from the leftover media from 7/8 were performed, as well as a clean streaking of a sucessfull transformant (from the LB Cam plates), as there were a low number of transformanants on the plates.

9/8/2018
Experiment: Gibson assembly of basic toolkit
Only one of the Mix 2 plates had colonies. We will continue to grow these plates. One colony was cleanstreaked and one was incubated in an overnight culture. We will retry the Gibson assembly. This time we will start with 70 ng backbone and work from there.
We increased the incubation time to ~4 hours to maximize our chances of a successful assembly
Experiment: Extraction of amilCP from distribution kit
The liquid O/N culture was miniprepped and subsequently concentrations were determined by nanodrop. This concludes the Extraction of amilCP from distribution kit experiment.

11/8/2018
Experiment: Gibson assembly of basic toolkit
Gibson assembly products was transformed and plated. Different amounts of cell culture were used in the hopes of getting more colonies. The first batch of gibson products had grown and exhibited a red color, reminiscent of RFP. AN overnight culture was miniprepped.

10/8/2018
Experiment: Investigation of unsuccessful Gibson assemblies
The attempted Gibson assemblies have thus far been unsuccessful yielding only red, RFP-expressing, colonies. This indicates either: i) wrong PCR amplification of the backbone from BBa_E1010 (A1 henceforth) or ii) that the assembly has not worked and that left-over BBa_E1010 is still present in the mix. Thus, A1 were amplified again according to the PCR protocol. This was then run on a 1% agarose gel together with the unsuccessful Gibson assemblies and BBa_E1010.
Conclusions from the gel:

1. The new PCR amplifications seems to have worked producing a clear bands, however some A1 seems to be present in the mix still, thus calling for more stringent cutting during gel purification.

2. The Gibson assembly have not been sucessfull at assembling the total fragment

3. A1 seems to present in the Gibson assembly - explaining the red transformants

Mycolab Overview

6/8/2018
Baking of trays: Tray from 26/7 baked for two hours at 100°C. Both trays from 25/7 and 26/7 baked at 70°C O/N.
Inoculation of boxes: As the mycelia has not been able to grow all the way to the bottom of the large boxes a hypothesis of air flow being the reason was raised. A box was supplyed with an air tube in the bottom of the box and inoculated as normally. Incubated at 28°C dark.

7/8/2018
Small bricks: An ice cube tray was inoculated with different ratios of rice and sawdust to see how that affected the texture of the bricks. See the detailed notebook on Tueday 7/8 for further details.

8/8/2018
Baking of trays:
Tray from 25/7: In the bricks made with overcooked jasmine rice a strong and tough air pocket had been formed. It was tough to break by force with tools. This pocket was filled with air. For the porrigde rice it was filled with liquid and much more wet that the overcooked jasmine rice.
Tray from 26/7: Dry ground rice from first tray seems to have grown well and is dry after baking. Second tray show less growth with many spores.
All from 25/7 taken out of tray and baked individually because htye were not yet dry. This is attributed to them being made from cooked rice already contained a high amount of water.

9/8/2018
We did more protoplasts according to the Protoplastation and Tranformation protocol, they were stored at -80°C.

10/8/2018
Small bricks: We wanted to test difference rice with sawdust. From earlier test (7/8) it seemed that 50% rice and 75% rice gave the best bricks. These ratios will be used with different kinds of rice. White rice, granulated rice, porridge rice, grounded porridge rice were used and one tray was with 50% and another was with 75%. See detailed notebook for Friday 10/8 for schematic of the trays. Incubated at 28°C dark.
Death test: Test from 3/8 showed no signs of mycelia growth.

Test from 8/8 showed signs of growth, although not mycelial. Plates put back in incubator for further inspection later. It seems like we are good with the baking. This means that the bricks that needed further drying should be dead as well - especially because they have had even longer in the oven than these tests.

Synthlab Overview

13/8/2018
Experiment: Gibson assembly of basic toolkit
After reviewing the available literature for troubleshooting Gibson assembly we have come to the conclusion that the problem probably arises from trying to assemble more than 5 fragments at once. Thus, we have decided to assemble the plasmid sequentially. Thus, Gibson assembly were performed of the TS1 homology regions, the pTrpC promoter and hph, the Hygromycin B resistance gene (This product referred to as TH) Additionally assembly of TS2 homology region and the CaMV terminator were performed (this product referred to as CT). The assembly were performed according to the Hi-Fi assembly protocol.

14/8/2018
Experiment: Gibson assembly of basic toolkit
Another round of Gibson were performed according to the specifications given 13/8. The resulting products from both rounds of assembly were amplified by PCR, utilizing primers a forward gibson-primer matching on end of the product and a reverse primer matching the other end, I.E. D0008, the forward primer for TS1 and D0009 the reverse primer for hph were used.
The gibson assembled fragments were then run on a 1% agarose gel (the first number specifies round and the second number specifies duplicate):

PCR products have been inserted following the headline

15/8/2018
Experiment: Geleletrophoresis of PCR produts of Gibson assembly
After yesterdays nice looking gelelectrophoresis, we wanted to do a new electrophoresis (replicate): This time we loaded 20 uL instead of 5 uL to get a sufficient amount of DNA for gel purification

PCR products have been inserted following the headline.
Given how vague the ladder is and how smeared the bands are, we assume the gel to be bad (given that is was also the very last drops of the gel bottle.
The second gel electrophoresis was run on machine RunOne #8. We loaded 25 uL of DNA. Based on online discussion we have identified overloading as the most likely problem
After the unsuccessful gelelectrophoresis, we realised that we would run out of PCR product of the 14/8 gibson assembly product after the next gel. For this reason we decided to make another 50 μL of TH1, TH2, CT1 and CT2. Due to the double banding the product, we decided to increased the cycle time to 5 min and 30 seconds (30 seconds is the minimum cycle, where 30 sec should be added for every kb. We added 5 mins).

16/8/2018
Experiment: Gel Electrophoresis of 15/8 PCR products
Due to the hypothesis that too high DNA concentrations were the cause of smearing in the two previous gel electrophoresis, we decided to reduce the loaded amount to 5 μL

18/8/2018
AmilCP PCR products.
11-12-21-22

PCR products have been inserted following the headline PCR composite 18/8
Amil, gfa, melA, Ao, HygB

PCR products have been inserted following the headline

19/8/2018
Tried to run gel for purification, unsuccessful, ran out of PCR products.

Mycolab Overview

14/8/2018
Death test: Test from 8/8: no sign of mycelial growth. Any growth present looks like bacteria or yeast.

Baking of trays: Ice cube tray from 7/8 ready for baking. Put in for baking at 100°C for four hours. Turned down to 70°C for O/N baking.
Boxes: Pipette boxes with tips for air checked: Only the rice/saw dust mix box had any noteworthy growth around the tips but not much growth in the bottom of the box.

15/8/2018
Baking of trays: Bricks taken out and put in sterile pipette boxes for storage. Both completely sawdust and completely rice were not coherent. The mixes became more coherent the more rice there was.
Boxes: Drylab made new boxes.

17/8/2018
Checked pipette boxes and ice trays. Rice + saw dust and rice + coffee should be baked in three days. All of them lacked strength which means no coherent results.
Boxes: Drylab made new boxes.

Synthlab Overview

21/8/2018
Experiment: 3A of genes w. camV Terminator
The following genes, terminator and backbone have been digested:
MelA, MelA - A.oryzae, GfaA, amilCP, HygB
The CaMV 35S terminator
A1 backbone

Of these, the amilCP, CaMV 35S terminator and A1 came as pladsmids. The others were linearized fragments from IDT. The resulting digestion mix had a concentration of 10 ng/µl. We used the ligation protocol to ligate the induvidual genes toghether with the backbone and the terminator

22/8/2018
Experiment: 3A of genes w. camV Terminator
Ligation: melA,Ao,gfa,amil,hph.
Transformation: The ligations from yesterday and the ligations from the failed 3A PCR experiment were transformed into E. coli and placed at 37 C overnight.
Colony PCR: In parallel, we tested the colony PCR protocol. It seemed like diluting the colonies doesn't give good results.

23/8/2018
Experiment: 3A of genes w. camV Terminator:
The transformants was a success.
Colony PCR: 3 colonies from each plate was suspended in water. A small fraction of this suspension was used for a liquid overnight culture, that can be used for a miniprep. The rest was heat-treated for 10 min and used for a colony PCR afterwards. The results show that the ligation products have the correct length.
Experiment: Second round of colony PCR (multicolony PCR) Explanation of procedure. 5 transformants plated on a new plate - and further transferred to the same tube.
Two PCR reactions - one using VR/VF primers, the other using [Gene]_fw/CamV_Term_bw primers.
Backup experiments: As a backup, we digested MelA, MelA - A.oryzae, Hph and GfaA and prepaired the ligation.
New experiment: 3A of AmilCP+CamV Terminator with. promoters
AmilCP digestion: Digestion of: Pgpd, PtrpC, PcamV, amilCP and A1
AmilCP ligation: Ligated with Pgpd, PtrpC and PcamV

24/8/2018
Experiment: 3A of genes w. camV Terminator
Created gel of multicolony PCR. In this experiment the forward sequencing primers were used together with the backward sequencing primer of CamV. It was conducted to test whether a multicolony PCR approach would work.
Backup Experiments: The backup genes were ligated and transformed into E. coli. The colonies were plated and left overnight. Delivered assembly: We have recieved an assembly from IDT cocsisting of 'pTrpC-HygB-CaMV 35S term'. This assembly was digested and ligated to be transformed tomorrow. When the gene is ready and amplified, it can be handed over to Mycolab for transformation into A.oryzae and G. lucidum.
Experiment: 3A of AmilCP+CamV Terminator with. promoters
Assembly of amilCP and promoters. The successful ligation and colony PCR of the 'amilCP-CaMV 35S term' assembly was digested and ligated with 3 different promoters: 'pGpd', 'pTrpC' and 'pCaMV 35S'. The ligation will be transformed tomorrow

25/8/2018
Experiment: 3A of AmilCP+CamV Terminator
Colony PCR of backup: The colony PCR was not successful. It is not clear why the gel showed no significant answers. The problems could include a failed ligation, failed PCR, failed gel or some other factor.
PCR amplification of delivered assembly: The delivered assembly was amplified with verification primers. The product has run on a gel and the results was mediocre (at best). The amplification is probably a success, but it cannot be confirmed from the gel.
Experiment: 3A of AmilCP+CamV Terminator with. promoters
Tranformation: The amilCP + promoter assemblies and the delicered assembly has been transformed and plated on cam plates

26/8/2018
Experiment: 3A of AmilCP+CamV Terminator with. promoters
The different transformants (amilCP + promoter and Hph marker) were colony PCR'ed.

Mycolab Overview

20/8/2018
Baking of bricks: Bricks baked last weeked seemed to still be alive; they were put back into the oven at 70°C. Baking the pipette box of rice + saw dust; mycelia can be seen everywhere. It has a strong and spongy strucutre so far. O/N 70°C.
Spore suspension: A new spore suspension was made from plates from 060818.

21/8/2018
Protoplastation and transformation: Inoculated plates containing Hygromycin B to check if the concentration of Hygromycin B is enough too inhibit growth.
Baking of bricks: Small bricks taken out and put back in containers. Large brick put back for more baking. Small part of large brick taken out.
Reinoculation of plates: New plates were inoculated and incubated at 4°C to assess growth at low temperatures.
Small bricks: Drylab made more bricks.

22/8/2018
Baking of trays: Drylab baked trays.
Protoplastation and transformation: Hygromycin B plates available will not work. A. oryzae not inhibited by hygromycin B. We try different approaches.

23/8/2018
Protoplastation and transformation: We try NTC as selection antibiotic instead. We try inoculating plates with different concentrations: 100 µg/ml, 200µg/ml, 400µg/ml, 600µg/ml and 800µg/ml. Could also try an in-house auxotroph of A. oryzae.

24/8/2018
Protoplastation and transformation: Growth on all plates with NTC although the higher concentration of NTC in media, the slower growth rate. Ganoderma received from Ecovative.