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 exciting 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.

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 modeling)

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

We used different PDA and MEA media for the species. 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.

More details here

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 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
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 protocol. Using part 1 and 2 from the BioBrick tutorial (Later adapted together with the resuspension 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 the 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 "top TS2" and "bottom TS2" 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 TS2b. 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.

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 . 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
amilCP, GfaA, MelA, A0, 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.

22/8/2018
Experiment: 3A of genes w. camV Terminator
Ligation: MelA,A0,GfaA,amilCP,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 using 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: pGpdA, pTrpC, PcamV, amilCP and A1
amilCP ligation: Ligated with pGpdA, 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: 'pGpdA', '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.

Synthlab Overview

27/8/2018
Experiment: 3A of genes w. camV Terminator
The colony PCR products was examined by gel elecroforesis and the following amilCP assemblies was identified as successful:
G3 (or G4 & G7), C3, C5

Furthermore, we chose H2 and H4 to be an overnight culture. We chose G8 as a control.

The colonies were cultivated further in a liquid overnight cultre for miniprepping tomorrow. Lastly, we redid the gel for the hph selection marker. From this, it seems like H2 is the most viable selection marker.

Based on these gels, we isolated specific cultures.

It seems like the backup digestion and ligation does not work. We will therefore redo that experiment, this time with linear inserts. We think that if the inserts excist as plasmids, there is a posibility that they can aquire only the terminator and not the combined ligation. Therefore, only linear fragments will be used. This ensures that a colony is either;
1) red and contains the RFP gene or
2) white and contains the correctly assembled plasmid.

28/8/2018
Experiment: 3A assembly of CaMV terminator onto genes of interest
The CaMV terminator was digested as downstream part, while the genes of interest, namely: GfaA, MelA and MelA optimized for A. oryzae were digested as upstream part. They were inserted into pSC1C3 (the RFP expressing variant was used for ease of identifying background transformants).

Experiment: 3A assembly of hph selection marker and amilCP constructs (containing)
The amilCP constructs containing promoter and terminator as determined by the cPCR from 26-27/8 were digested as downstream part, while the hph selection marker from IDT were digested as upstream part. pSC1C3 was used as backbone.

29/8/2018
Experiment: 3A of genes w. camV Terminator:
Based on the Gel Tuesday, we isolated the C3, C5, H2, H4, G3, G4, G7 and G8 cultures, I wanted to confirm the construct we have made. Given the differing migration lengths from the 27/8 gel electrophoresis, we are currrently not sure whether it is the C1 or the C3 that is the successful assembly of P. CaMV-amilCP-CaMV term or whether any of them actually are. Likewise for G3, G4 and G7.
In this experiment I am to verify the sequences by amplifying the gene regions overlapping two adjacent gene fragments.Tomorrow the products from this PCR will be run on a gel, to hopefully either verify or disprove the existance of expxected gene fragments.
The PCR used a ONETAQ polymerase. The PCR was run with an anealing temperature of 49C and a elongation time of 1 min.

30/8/2018
Experiment: 3A of genes w. camV Terminator
Today we performed gel electrophoresis on the 29/9 verification PCR.

Experiment: Gibson assembly of gene cassettes
PCR extention with homology regions to allow for Gibson assembly were performed on the genes of interest. This was done using specific primers and Q5 PCR.
After the PCR the products were run on a gel. The lanes corresponds to the above chart in terms of product (So lane 1 contaings pTrpC with homology regions for MelA and so forth):

31/8/2018
The following genes, with their respective Gibson overhangs, were purified using the gel extraction protocol as they were the only sucessfull PCR amplified genes:

• pTrpC (MelA)
• pTrpC (GfaA)
• MelA
• MelA (A. oryzae)
• GfaA
• CaMV terminator (MelA)
• CaMV terminator (GfaA)

Experiment: 3A assembly of hph selection marker and amilCP constructs (containing)
Transformants containing the 3A assembled hph selection marker were screened for the correct inserts by cPCR with the verification primers, VF2 and VR. 5 Colonies were chosen from each of the CaMV promoter containing transformants and 8 colonies were chosen from the pGpdA containin transformants, due to higher background (estimated by amount of RFP-expressing cells). A one-taq PCR amplification were then performed and the products run on a gel:

Based on the gel none of the screened mutants contained the right insert, which should be about 3200 bp, though g34 seem to containg some sort of additional assembly. Thus, this should be investigated.

Mycolab Overview

27/8/2018
Protoplastation and transformation
Transformation media prepared. See specifications in the protocol for fungi media.

Statistical study: Tray 2, 7, 8, 6 were placed into the oven.

28/8/2018
Statistical study: Tray 6 and 8 were removed from oven.

Protoplastation and transformation
Plates with different concentrations of NTC prepared: 200, 400 and 800 and a positive. E. coli O/N cultures prepared with strains for MiniPrep.

29/8/2018
Protoplastation and transformation: Mini-prepped E. coli cultures from yesterday according to the QIAprep Spin Miniprep protocol and nanodropped the minipreps.

30/8/2018
Protoplastation and transformation: Transforming mini-prep into protoplasts to test protoplasts and miniprep. 5 transformations in 5 tubes. Each plated onto different plates with different NTC concentrations. Using protoplasts from 9/8.

Statistical study: Tray 1 and trayy 5 placed in oven at 70°C. Tray 2 and 6 placed in oven at 100°C.

Synthlab Overview

3/9/2018
Experiment: Verification of PCR amplified camV terminator
The camV. T. PCR amplificants were run on a gel to verify that the amplification had been successful. It seems that the amplificants seem successful.

The colonies were cultivated further in a liquid overnight cultre for miniprepping tomorrow. Lastly, we redid the gel for the hph selection marker. From this, it seems like H2 is the most viable selection marker.

Transformation of all the ligates from today were transformed into Dh5-α-competent cells following the 3A transformation protocol
Experiment: Gibson Assembly of gene casettes.

Since the previous PCR amplification with Gibson primers didn't yield any backbone to assemble into, as well as lacking some other amplifications, these were attempted again. This time both plasmid-inserted and linearized PCR amplification was performed. As can be seen from the gel below the plasmid amplifed backbone (PCR product 4 in the freezer) yielded an amplfied backbone.
The products ampflied are pTrpC-MelA (A. oryzae), GfaA, CaMV-GfaA, backbone from plasmid and backbone. The last three are from an investigation of a construct from the hph-selection marker 3a assembly:

4/9/2018
Experiment: Gibson Assembly of gene cassettes
As seen on the gel picture from Monday (September 3rd), we have two clear bands. These were gel extracted using the protocol. The resulting DNA concentrations are 38.1 and 38.6 ng/µl respectivly for band.

Experiment: Colony PCR
5 successful transformants from each plate was extracted and used for a colony PCR following the colony PCR protocol. We used the verification primers for this PCR to examine if length of the transformed constructs. This will be used to verify the digestion and ligation. In parallel, we experimented with an alternative technique for colony PCR. We scraped multiple colonies from the same plate off and treated it as if it was a colony. By doing it this way, we hope that we can screen plates faster, as we are looking for a band of the correct size in the gel. A possible limitation of using this technique with the verification primers is that other plasmids, such as the RFP will show bright bands, while our gene of interest might be very dim. This can be avoided by using other primers, but we will discuss this at a later point.

5/9/2018
Experiment: Gel results of colony PCR
The colony PCR from 4/9 was run on a gel and gave rather dissapointing results

Troubleshooting:

• Premixing a 'master-mix' might be problematic?
• The annealing temperature was too high
• The extension time too long
• Too long amplification sequences
• An error was made mixing the master mixes for the colony PCR
• The lysated contain insuficient DNA or otherwise make for a bad template

PCR to test the lysates:
We have made a small PCR to test the viability of the lysates as template. In this PCR we have the colony lysates 1,2,3, a positive control using RFP (0023) and a negative control. We have reduced the annealing temperature to 49*C and otherwise we have tried to keep the parameters the same. Should we get good results, we will retry the colony PCR using the lysates and the new conditions. Should the results fail us, we can consider to try to use Q5 PCR on the lysates or make a new batch.

6/9/2018
Gel results from the lysate PCR test: The PCR results from the experiment on the 5/9 were run on a gel. We can see that there are some results, which suggests the lysates are not the problem. This could mean that it was the annealing temperature which was too high, that was causing the problems.
9/9/2018
Experiment: Colony PCR of transformants.
A colony PCR of the Gibson transformants was prepared, with 5 colonies picked from each plate. This was done early in the day, which may not have allowed the RFP-expressing tranformants to show yet, thus increasing risk of picking a wrong transformant. A one Taq-PCR was performed with verificiation primers at 53 degrees celcius.

Mycolab Overview

3/9/2018
Protoplastation and transformation
Transformation plates had already sporulated. They were replica plated onto minimum media plates each containing the same NTC concentration as the original transformation plate.

5/9/2018
Protoplastation and transformation
Replicate plates phtographed and put in fridge. A. oryzae plates incubated at 20°C.

7/9/2018
Protoplastation and transformation
Ganoderma resinaceum protoplastation experiments started according to protocol. For incubation until Wednesday 12/9. 20°C A. oryzae plates checked. There is growth but it is very little.

9/9/2018
Protoplastation and transformation.
Transformation of colour genes according to protocol.

Synthlab Overview

10/9/2018
Experiment: Gibson Assembly of gene casettes
After two days in the cabinet incubator, the red colonies (RFP) were easily distinguishable from the white colonies (maybe succesful transformants). In total 12 colonies were to be found of the 3 agar plates, and these were isolated for a second round of colony PCR on the gibson transformants, Gel of 1st and 2nd round of colony PCT:

The colony PCR of the white colonies from the 10th of september (today) all seem to be succesful gibson transformants. Colony 1-4 have been prepared as an O/N culture for miniprep tomorrow.

11/9/2018
Experiment: Gibson Assembly of gene cassettes.
Colony 1-4 had been grown O/N and 0.5 ml was transfered to a glycerol stock and placed at -80 C. The rest of the O/N culture was miniprepped. The resulting concentration of plasmids can be seen here. Plasmid 0063 has been sent to sequencing. Experiment: 3A assembly of composite parts.
We PCR amplified sequnces of 0056, 0057 and 0058 to increase their concentration for sequencing. In addition, we have prepaired an O/N culture of 0056, 0057 and 0058, but it is not certain we will need them. The plasmids have been sent to sequencing to be verified.

12/9/2018
Experiment: 3A assembly of NTC resistance gene
As the hph selection marker may not be a viable option in A. oryzae, we have decided to trying NTC as a selection marker. The Nat1 gene has been PCR amplified to contain the BioBrick prefix and suffix in a previous experiment. Now we will assemble it into a plasmid with a promoter and terminator. As with a lot of the consructs, we are testing different promoters and we will also be doing that here.

Experiment: Digestion.
We started with assembling the Nat1 gene together with PcamV (0028), pTrpC (0018) or pGpdA (0008). We are using RFP (0024) as the backbone. The reason for starting with the promoter, is that this unfinished construct could have some limited use in the finished product, while the gene and terminator has no direct application.

Experiment: Gibson Overhang Extension PCR
Homology regions for Gibson assembly was added via PCR extensions to specific fragments. This time the 5x round of the PCR were done at the intial annealing temperature of the primers and afterwards the annealing temperature was increased to account for the elongated DNA molecules present which can then bind the full primer (with the extension on). The products was run on a 1% agarose gel producing the following bands. Thus, all fragments was succesfully extended except for GfaA, which continues to be a problem. This could very well indicate a need for redesign of primers.

Mycolab Overview

10/9/2018
Protoplastation and transformation.
Performed another transformation of colour genes. The protocol for protoplastation of the A. oryzae was initialised.

12/9/2018
Protoplastation and transformation
New spore suspension. A. oryzae protoplastation according to protocol. Next Ganoderma protoplast step taken. There was a human error and the samples were centrifuged instead of vortexed.

14/9/2018
Protoplastation and transformation
Osmotic buffer and sorbitol solution made and autoclaved. This is the day that the Ganoderma protocol was attempted changed for the first time. With no Driselase available we had to think of other ways to protoplast Ganoderma. After consulting Ecovative we tried the first time with higher concentrations of Glucanex instead of both Glucanex and Driselase as originally in the protocol.

Synthlab Overview

18/9/2018
Experiment: 3A assembly of NTC resistance gene
The plasmids containing a promoter and the Nat1 gene were PCR amplified using verification primers and run on a gel to ensure a correct ligation.

These results look promising and we will continue with these plasmids. We are using the first of the four plasmids from each promoter construct.
The digestions were ligated and will be ready for transformation tomorrow.

Experiment: Gibson Assembly.
The Gibson products from 17/9 were transformed, plated and incubated at 37 C O/N.

Experiment: Existential Crisis
Have you ever considered that the parking lots built in your lifetime will still be there, when you are long gone and forgotten?

19/9/2018
Experiment:3A assembly of NTC resistance gene
The ligated products were transformed into E.coli and plated on LB cam plates.

Experiment: Gibson Assembly
The plated colonies may have been contaminated. We are solving this in two ways. We have redone the transformaiton and plated everything again We have spred the liquid culture on another set of plates.

Experiment: Gel of PCR amplified gene fragments for Gibson.
The PCR amplified gene fragments for gibson from yesterday (18/9) were run on a gel. The resulting gel:

Given this result, samples 3, 4 & 5 (The three pTrpCs with the right prefix) were isolated for gel extraction (performed 21/9).

Experiment: Preparation of amilCP for mycolab
For the transformaiton mycolab needs a steady supply of amilCP. We will provide that by producing multiple O/N cultures and puryfing the plasmids. We are also interested in discovering if the transformation is easier with linear DNA or with plasmids, so we will provide both. Multiple O/N cultures of C3 and G3 (See week 35) have been made from a glycerol stock.

21/9/2018
Experiment: 3A assembly of NTC resistance gen and Gibson Assembly
The colony PCR products were run on a gel. The results were rather disappointing.

Test of the colony PCR lysates: Due to the dissapointing results from the previous PCR, we wanted to test whehter the lysates were the problem, for this reason we selected the following lysates as a probe: F1 (RFP contaminated?), F2, C1, C2 and a positive control POS (RFP plasmid 0024).

22/9/2018
Experiment: 3A assembly of NTC resistance gene and Gibson Assembly.

The only interesting results from this Gel electrophoresis were C4,D1-D4,E2-E4.
The cultures for C4, (D2, D3?), E2, E3 where set to incubate O/N.

• C: pCaMV+Nat1+CamC Term
• D: pTrpC+Nat1+CamC Term
• E: pGpdA+Nat1+CamC Term

Given the migration patterns on the gel, I suspect that the the C,D,E constructs are too short to have both the promoter, the NAT1 gene and the terminator. This can be investigated from the O/N cultures.
Concluding remarks: It seems that this round of gibson transformations was unsuccessful.

Experiment: PCR on the A. oryzae Lysates.
The PCR on the A. oryzae lysates didn't yield any good results. 23/9/2018
Experiment: Tranformation of A. oryzae with amilCP.
A. oryzae was tranformed with amilCP cassettes, either containing the pCaMV promoter or the pGpdA promoter. Both cirucular and linear versions of the PcamV promoter constructs were used. 4 replicates of each was made and placed at 28 degrees celicus for growth.

Mycolab Overview

17/9/2018
Protoplastation and transformation
Ganoderma protoplastation: No Driselase found anywhere in the lab so trying out with different concentraions of Glucanex:

• 20 mg/ml
• 30 mg/ml
• 40 mg/ml

The 40 mg/ml is the same concentration as in the Aspergillus oryzae protocol which is what prior knowledge we had to draw from. Transformation with chromoprotein: No colour colonies which would indicate that there has been no transformation. But! The transformed spores are very green compared to the yellow-green spores of the wildtype which indicates some change.

18/9/2018
Protoplastation and transformation
First part of the process for isolating protoplast done. Tubes labelle 1, 2 and 3 put in the fridge for finishing up the next day.

19/9/2018
Protoplastation and transformation
Samples were centrifuged but no pellet was isolated. Samples were thrown out and the protoplasting and transformation protocol was replanned.

20/9/2018
Protoplastation and transformation
Inoculated 15 PDA plates with A. oryzae. Ecovative Ganoderma protoplastation protocol restarted. Next step is on Tuesday.

21/9/2018
Protoplastation and transformation
Transformation of PcamV and PgdgA of A. oryzae.

23/9/2018
Protoplastation and transformation
Transformation of A. oryzae with circular and linear fragments. Used 10 µl for all transformations.

Synthlab Overview

24/9/2018
Experiment: PCR amplification of amilCP constructs (for transformation).
Anealing temperature of verification primers: 53 Celsius. We are amplifying pGdp-amilCP-CaMV and pCaMV-amilCP-CaMV which have a length of ~1600 bases and 1300 bases. We are running the PCR triplets. We are using 1 µl og DNA for each reaction. In total, we have 8 amilCP reactions, a positive with RFP and a negative.

Experiment: Prepartion of RFP in pSCB1A3.
RFP was aquired from the kit and transformed into E. coli and plated on ampicillin-containing plates.

Experiment: Miniprep of G3 and C3.
4 cultures of each plasmid was miniprepped. They still need to be nano-dropped.

25/9/2018>
Experiment: Melanin 3A assembly.
We are very interested in testing our MelA and MelA-A.oryzae constructs work in a fungi. We will therefore try to assemble them using 3A assembly again.

Experiment: Purification of promoters for E.coli
BBa_J23119 is a strong constittutive promoter for E.coli from the Anderson promoter library. We want a promoter for E.coli to quickly verify that our MelA genes. We will be using the brick in two different backbones, pSB1C3 and pSB1A2. These part are found on the distribution kit.
The following parts have been transformed into E.coli and plated on their respective plates:

• BBa_J23119 - pSB1C3: Plate 3, well 17O
• BBa_J23119 - pSB1A2: Plate 4, well 17B

26/9/2018
Experiment: Purification of promoters for E.coli.
A 17O culture has been transfered to LB cam liquid media and is being incubated O/N. The 17B transformants where wrongfully plates (as these contain the Amp resistance), but a new LB amp plate was prepared and the 17E cultures replated and let to incubate O/N.

Experiment: Melanin 3A assembly.
The amp resitance backbone pSB1A3 where minipreped and digested as backbone for the assembly.

27/9/2018
Experiment: Melanin 3A assembly.
The ligations were transformed into E.coli and plated on LB amp plates. RFP (0025) was used for as a positive control and was plated on a LB cam plate.

28/9/2018
Experiment: Melanin 3A assembly.
Onetaq protocol using verification primers and a annealing time of 3 minutes per cylce.

30/9/2018
Experiment: Gibson fragments.
The fragments intended for Gibson assembly showed terrible results on the gel. We need to retry the PCR to get usable results.

Experiment: Melanin 3A assembly.
Some of the colonies were transfered to an O/N culture and we can hopefully extract successful plasmids tomorrow.

Mycolab Overview

24/9/2018
Protoplastation and transformation.
Samples from transformatinos (21/09/18) taken for colony PCR. See synthlab notebook from this week for rest of this. A. oryzae protoplastation initialized.

25/9/2018
Protoplastation and transformation.
No PDB available, so the Ganoderma protocol was postponed to tomorrow to make the PDB.

26/9/2018
Protoplastation and transformation.
Funnels prepared for protoplastation of A. oryzae. Digestion of A. oryzae was postponed to tomorrow. So the first couple of steps were done and then put in the fridge to be finished tomorrow. First steps of Ganoderma protocol carried out as well.

27/9/2018
Ganoderma protocol continued. This time with varying concentrations of Glucanex, shaking speeds during digestion and digestion times. For details on this, see the protocol for protoplastation of Ganoderma. No cell strainers are available so the samples that were only given 2-3 hours of digestion were not filtered correctly. Henceforth sterile funnels with miracloth were used instead. We continued working on the protoplastation and transformation of A. oryzae.

28/9/2018
Ganoderma protoplastation of samples that were given 19-24 hours of digestion continued. Following the protocol, all samples (Both 8 19-24 hour and 8 2-3 hour samples) were put into the fridge until Sunday for finalization of the protocol.

30/9/2018
Finalised Ganoderma protocol by centrifuging. No pellet, so no protoplast. Back to drawing board.

Synthlab Overview

1/10/2018
Experiment: Amplified Insert Assembly of MelA.
The promoter-MelA assemblies seem to be successfully assembled, so we will try to add a terminator to the constructs.
We are using 0113-0116 as the MelA parts and 0014 was used as the terminator and backbone. We adapted the 3A assembly protocol to just using digestion of the parts and then ligating them. If this doesn't work, we may have to try using PCR amplification to produce enough insert.The products were ligated using the 3A protocol with the minor adjustment of just using two parts instead of three. The ligated DNA were then transformed into E.coli and plated 150 µl on LB cam plates.

Experiment: PCR verification of NAT1 and Beta-tubulin in A. oryzae.
A Onetaq PCR was performed on the A. oryzae lysates PC1, PC2, PC3, PC4, PG1, PG2, PG3 and PG4 to verify the presence of NAT1 and Beta-tubulin. Each of the lysate samples were amplified using the Seq_NAT1_in_fw/Seq_NAT1_in_bw or seqP_betatub_in_fw/seqP_betatub_in_bw primers. The resulting gels were dissapointing and the lysates declared unsuccesful End of Experiment: PCR verification of NAT1 and Beta-tubulin in A. oryzae.

Experiment: Sequencing of biobricks.
Sequencing results:

• Confirmed the 0092,0093,0094 plasmids.
• Showed that 0101,0102,0103 does not contain NAT1 nor terminator. Only the promoters were present in the plasmids.

3/10/2018
Experiment: Amplified Insert Assembly of MelA.
The plated cultures have grown overnight and 3 plates showed growth. We have therefore made liquid O/N cultures of 0113, 0114 and 0116. The plates continues to incubate at 37 degrees.

Experiment: Melanin 3A assembly.
Two cultures that showed positive results on the gel were grown O/N and miniprepped. They have been added to the spredsheet as 0127 and 0128.

Experiment: Sequencing of biobricks.
Some of our promoter-melanin constructs were sent for sequencing. We hope to determine if it is worth it to continue with this line of experiments, and we will therefore verify that we have the correct constructs.

4/10/2018
Experiment: Amplified Insert Assembly of MelA.

Experiment: Assembly of Nat1 plasmid.
The Nat1 resistence gene has to be inserted into a backbone to be submitted to the registry. We will therefore cut it out and insert it into the backbone of RFP (0023).
The Nat1 gene, with the prefix and suffix, and RFP (0023) was digestied with EcoRI and PstI. Following that they were ligated and transformed into E.coli.

Experiment: Sequencing of BioBricks.
The miniprepped promoter-MelA-terminator constructs were sent for sequencing.

5/10/2018
Experiment: Assembly of Nat1 plasmid.
The plated colonies didn't look great, so we chose the least red colonies and ran them on a colony PCR.
In parallel, since the results seemed very poor, we have redone the digestion and ligation, but this time we are using other enzymes. We suspect that PstI may not be able to cut our Nat1 fragment, as our primer design hadn't allowed for extra bases following the cut site. We therefore used EcoRI and SpeI as our enzymes. In addition to using other enzymes, we also tried to use a linearized backbone from the distribution kit to see if that could increase the success-rate.

Experiment: Colony PCR on overnight cultures.
A total of 4 white colonies (N1,N2,N31 & N32) were discovered on the three plates for NAT1 transformants. A standard verification primer Onetaq was performed on these cultures and they were run on a gel.

Suprisingly, cultures N31 and N32 seem rather interesting and should be grown O/N.

6/10/2018
Experiment: Colony PCR of the overnight.
A colony PCR was performed on the overnight NAT1 transformants. The PCR used was a standard verification primer PCR with 60 seconds extension time.

None on the colonies seem promising and looks mostly like selfligated backbones.
Experiment: PCR on N31 and N32.
A PCR with primers was performed on the N31 and N32 lysates from yesterday (5/10) using the primers.

Suprising something worked. However the results a both encouraging and deconsolate, as the two first samples shows promising 400 bp bands, however the negative test gives more or less a band in the same height, for which reason I believe we should run another gel tomorrow.

7/10/2018
Experiment: Rerun of PCR on N31 and N32.
The verification PCR from yesterday was repeated using 51 C as annealing temperature instead of 64*C. The results are like those of yesterday and sadly rather inconclusive.

Experiment: Digestion, Ligation & Transformation of NAT1.
A new digestion of NAT1 was performed using EcoRI & PstI was made. Each of the NAT1 digests (N1,N2,N3) were ligated with both a digested backbone and a linear backbone resulting in the six ligates (N1B,N1L,N2B,N2L,N3B,N3L). These we subsequently transformed and plated alongside with the gibson transformants. Experiment: Gibson assembly of NAT1 into backbone.
Using the prefix/suffix homologous ends of the linear backbone and our NAT1 PCR extracts, we performed gibson assembly for insertion of the NAT1 gene into the backbone. These were transformed and plated for growth O/N.

Experiment: Overnight cultures of NAT1.
Two addtional O/N culture of N31 and N32 were prepared in liquied LB cam media and left of incubate.

Mycolab Overview

1/10/2018
Ganoderma protocol started.
Ganoderma was inoculated on PDA plates and incubated at 30°C.

3/10/2018
We inoculated 12 plates of A. oryzae on PDA and incubated them at 28°C and prepared sorbitol solution and funnels for the aforementioned Ganoderma protocol.

5/10/2018
Ganoderma protocol continued. This time with twice as many samples to try an up the amount of mycelium in the tubes. 1 ml og PDB was put into 32 tubes and inoculated with Ganoderma. Incubated at 30°C and at either 85 rpm and 150 rpm for 48 hours instead of 24, again to get more mycelium. Protoplastation of A. oryzae continued.

7/10/2018
Ganoderma protocol continued. The digest and start of isolation were done for the samples that were only given 2-3 hours and the digestion was started for the samples that need 19-24 hours.

Synthlab Overview

8/10/2018
Colony PCR and Nat1 transformants were run. a miniprep of N31 and N32 was performed.

We had failed miniprep of A4 and A5 melA constructs as the A4 and A5 were incubated in media with a wrong antibiotic.

10/10/2018
Miniprep of O/N cultures was performed and samples were sent to sequencing.

11/10/2018
Results of the sequencing showed that all nat1 attempts have been unsuccessful.

14/10/2018
Experiment: PCR Verification of A. oryzae transformants.
New gDNA was purified from 7 different A. oryzae plates. 4 plates contains a (probably) CAC from linear DNA transformation, There are 2 transformants from a GAC transformation and 1 MelA transformant.

Experiment: Transformation of A. oryzae with melA genes.
A. oryzae protoplasts were transformed with pcamV-melA-tcamv constructs (0131) and (0130) and left to incubated on transformations media containing 0,6 mg/mL tyrosine.

Mycolab Overview

8/10/2018
Ganoderma protocol continued: The 19-24 hours samples taken out and started the isolation procedure. Put in fridge with the other samples.

9/10/2018
Looked at transformation plates with Kyle to see if something might have transformed: He thinks that the media being coloured and the spores looking greener than usual on the transformants might indicate that something has been transformed. New bricks were made to see if they secrete the colour into the bricks.

10/10/2018
The Ganoderma protocol was finished by centrifuging for 10 minutes at 10000 rpm. There was no pellet.
Liquid cultures of the suspected transformants were inoculated and incubated at 28°C and 150 rpm. The cultures were inoculated with both green spores and white spores to see if they secreted any kind of colour.

11/10/2018
As a last try, the Ganoderma protocol was restarted, this time with driselase as we hand received some the same day. There was no time to make new plates to incubate for 5 days, so we had to try with some of the old plates. Three tubes with 1 ml PDB was inoculated for continuation the next day.

12/10/2018
The three tubes were taken through the digestion steps and put in the incubator at 30°C, 85 rpm.

13/10/2018
The samples were taken out of the incubator. The incubator had been changed to 28°C, 100 rpm when we returned to the samples. The samples were run through the first steps for isolation of protoplasts.

14/10/2018
Samples were centrifuged according to protocol but there was no pellet.

Synthlab Overview

15/10/2018
Experiment:PCR Verification of A. oryzae transformants.
The purified gDNA was PCR amplified using the AmilCO Forward primer and the tCaMV Backwards primer. This should reveal bands around 230 bp. (0092) was used as a positive control.

We see bands around 200 bp, but it is not certain if this is our gene of interest or not.

We ran the PCR products on a 2% agerose gel and we got the following:

Here it could seem like we have a band on the first sample that matches the positive control. Another PCR was made to verify the results, but this showed signs of contamination, as the negative also showed bands that was alsom identical to the positive.

16/10/2018
Experiment: Confirmation of amilCP construct integration.
Another round gDNA preparation and PCR was performed to confirm integration of amilCP constructs in apparent transformants. Extra care was taken in order to ensure no leakage from wells as this had been a problem earlier. These gel can be seen in the results page for amilCP.

17/10/2018
Experiment: Sequencing of gDNA.
We would like to know where the inserted fragments are in the genome o A. oryzae. Therefore, we have send a sample of the most successful transformants to sequencing with primers that point out of the cassette.
We tried to ethanol percipitate our previous samples, but we got very low DNA concentrations, which means we cannot expect resonable results. We have therefore performed a new extraction of gDNA from the same spot on the plate and disolved the DNA in 50, 100 and 200 µl of Milli-Q water.

Team Overview

The DTU BioBuilders are ready and thrilled for Boston. Good luck to everyone.