Team:DTU-Denmark/Notebook
Week 5 (Jan 29 - Feb 4)
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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.
Week 15 (Apr 9 - Apr 15)
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Team Overview
The annual BioBrick Tutorial was held and 89 exited and talented iGEM participants were gathered at DTU for an exciting weekend.
Week 17 (Apr 23 - Apr 29)
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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.
Week 18 (Apr 30 - May 6)
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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.
Week 23 (June 4 - June 10)
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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.
Week 24 (June 11 - June 17)
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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:
To illustrate an examine the growth rate (for modelling)
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.
All media was prepared before 11 am to be autoclaved. The actual weights of the different media was:
MEA Media Materials Weight (g) Weight (g) Weight (g) Malt extract 47.6 49.9 52.5 ZnSO4·7H2O 0.01 0.012 0.01 CuSO4·5H2O 0.05 0.049 0.051 Agar 14.99 14.99 15.04 PDB Media Potato dextrose extract Weight (g) Weight (g) Weight (g) Malt extract 19.54 39.12 58.56 ZnSO4·7H2O 0.012 0.012 0.0107 CuSO4·5H2O 0.0056 0.0053 0.0049 Agar 15.02 15.04 15.04 All media were stirred with a magnetic stirrer at around 110 degrees Celsius and 1000 rpm until homologous.
The species available (Pleuratus ostreatus) was inoculated on the solid PDA and MEA agar plates with a sterile toothpick under a fume hood and left at 30 degrees Celsius. It was on Monday discovered that the sample given was of mycelia without any spores and the process was redone.
Week 25 (June 18 - June 24)
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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.
Week 26 (June 25 - July 1)
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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.
Week 27 (July 2 - July 8)
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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. -
Drylab Overview
Up until this week, we have had a normal semester. Now all members, of drylab, are able to be working full time in the iGem project, not counting scheduled personal holidays. This week Mathies worked on Poisson HMM (Hidden Marcow model) and investigating binomial distributions with regards to simulating hyphal growth and branching probabilities. Nicolai is manufacturing/welding growth boxes for the Wetlab group. They need these to be able to make consistently shaped fungi squares for drylab compression testing. Lau and Mathias are investigating other modeling possibilities; how to grow Mycelium on different substrates, and how we, as a group, can enhance our productivity and usefulness for the other subgroups. Hannah collected a lot of data, to be used for more precise modeling parameters, and is working on writing a program that will be able to calculate growth speed using image recognition. Collectively we made a safety assessment and handed for review for us to be able to gain access to proper material characterizations machines at the mechanical department at DTU.
Week 28 (July 9 - July 15)
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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. -
Drylab Overview
We began the week with a meeting to set the course for the rest of the week. This week Nicolai is on holiday. On the modeling side of things, the work on the HMM is coming along nicely but it is not clear how it can be related to real life. The model outputs the probabilities of branching for individual hyphae. The bigger picture is not clear for this model. Hannah is working on codon optimization after having dropped the imaging programme. The reason being that the color variation of the sample pictures was too great and writing a program to be able to take this into consideration proved to be too hard. An alternative program was used to do this, called VGG Image Annotator (VIA) (http://www.robots.ox.ac.uk/~vgg/software/via/), where the radius of all the colonies were manually annotated. The area of each fungal colony was calculated and visualized in plots.
Mathias and Lau made good progress in investigating live imaging of Mycelia. The purpose is to nail down real parameters spaces for use in simulations. We handed in the safety assessment to DTU Byg (Mechanical Department) for review/verification and got a short tour of the material lab. Many different people were contacted in regards to material supply and modeling/simulation. More growth boxes were manufactured.
Week 29 (July 16 - July 22)
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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.
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Drylab Overview
This week Nicolai has come back. He assessed, based on the team's meetings with DTU Byg last week, that manufacturing metal inoculation boxes for Wetlab would become far too big of a task since it was unearthed that we would need over 100 boxes. It was decided that we order silicone ice-cube trays instead, both for us to focus on other pressing matters and for the consistency of the cubes. He also started work on a mission architecture including a design the brick for us to construct a martian hut. We obtained foam samples through a contact from last week. Hannah and Lau assisted the Myco-lab subgroup since they were struggling with their workload. At the end of the week, Nicolai began working on a DIY hydraulic press. We weren't going to be able to use the DTU byg machinery before a lab technician would return from his holiday in week 32. Mathias started researching if there existed a gene regulating the frequency of hyphal fusion in the fungi. This was based on findings from polymer science, which showed that vulcanized rubber showed increased strength if the network of rubber was more interconnected or more elastic if the rubber was less interconnected. This would be possible if we could find a homolog of a known gene that regulates hyphal fusion.
Week 30 (July 23 - July 29)
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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.
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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:Ground jasmine rice
Porridge rice
Ground porridge rice
Washing jasmine rice
Finely ground rice
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. -
Drylab Overview
This week Hannah and Lau were helping Myco-lab in growing “ice cube fungi”. They are being grown on a various substrates and inoculated at different times and temperatures. Mathias was on holiday. Nicolai was “fighting” with DTU skylab, as their technicians bailed on appointments, making the progression of the DIY hydraulic press slow and wasteful. Late friday, the small project was completed, only missing some teflon tape to seal the thread. Over the weekend Nicolai made a short video showing the manufacturing process. Mathies planned the data structures for the experimental setup for DTU byg. The purpose is to minimize the necessary test that have to be done, to save time and resources.
Week 31 (July 30 - August 5)
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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.
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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.
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Drylab Overview
This week Mathias returned and Hannah went on holiday. The DIY hydraulic press was completely finished and ready to use. Nicolai restarted the mission architecture and brick design. Mathias reached out to a polymer expert (Mika Torkkeli, Postdoc) to discuss the role of the network structure/interconnectedness in the strength of polymer networks, it was confirmed that the sparse mycelium network could perhaps be analogous to the network structure of vulcanized rubber. But because of the macroscale of the network and the sparseness of the network the effects might be less drastic than those found in rubber. Another challenge was that the only gene that was identified that might increase hyphal fusioning also affected mycelium density negatively. It was deemed to be too uncertain if we could control these effects away and whether or not the effect size would be worth it.
Week 32 (August 6 - August 12)
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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: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.
The Gibson assembly have not been sucessfull at assembling the total fragment
A1 seems to present in the Gibson assembly - explaining the red transformants
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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.
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Drylab Overview
6/8/2018
It was decided today that we are going to pick up the mycelium growth simulations, but approaching it differently than the previous attempt involving hidden markov models. It started out with a discussion about how a mycelium develops over time and discussing parameters based on experimental measurements, such as branching frequency and branching extension rates. Work was begun on two complementary growth models: a detailed micro growth model with a focus on hyphal growth and a meso growth model based on a system of partial differential equations describing the change in biomass density as a function of time, nutrients, space and species characteristics.
Nicolai and Lau collaborated in the sense that the brick design was finalized and Lau started simulating structures constructed using the brick design. Mathias and Nicolai tried to get a hold on the DTU Byg technician who could teach us to use their material properties machines. But he was still not available even though his schedule said otherwise. The first preliminary test was done using the “home built” hydraulic press. It initially showed a rough link between dryness and toughness. The more dry samples were generally more resistant to compression. 2 different kinds of foams were also tested to see if they were comparable to the material properties of the fungi. They were not, since they were actually tougher.
Week 33 (August 13 - August 19)
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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.
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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.
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Drylab Overview
13/8/2018
This week Mathias, Hannah, and Mathies were successful in completing some initial growth simulations, in which mycelium grew from one spore, using their new models. After these results, the code was optimized in order to simulate growth from multiple spores and see their progression. A gif, visualizing the growth using the micro model, was made and it was stunning. Nicolai and Mathias managed to get a hold of the DTU Byg technician and received training in the machinery in order to make really good compressional tests. The compression tests that our foam-like material is not really yielding the type of results we wanted e.g. we were expecting to see a clear “snap” when the sample was under sufficient compression. We are not seeing any “snap/crack”, only an increasing amount of compressional force, until the machine stage bottoms out. What we can do is to stop measuring at a given deformation, and look at HOW that acts. This is also taken into consideration for the simulation of the “hut design”. Since we have a finished design, it only needs to get realistic inputs. Lau applied physics (gravity, winds etc.) to the 3d model, but had problems with convergence.
Week 34 (August 20 - August 26)
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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.
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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.
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Drylab Overview
20/8/2018
Great steps have been taken since last week, where we now have an approach to estimating the density of the mycelium.
Still trouble with 3d convergence.
21/8/2018
Try to create much simpler model to just try out the software, now have trouble visualizing the deformation.
24/8/2018
Used the knowledge and confidence from simpler model to locate, that the error was in the visualization. Successfully created a test video, of a combination of 6 bricks(see drylab folder), created a design with round edges to allow rotation
Week 35 (August 27 - September 2)
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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.
GelDoc 2018-08-27 13hr 06min - C1 -C2 -C3-C4-C5-C6-C7-C8-C9-C10-H1-H2-H3-H4-NEG
GelDoc 2018-08-27 G1-G2-G3-G4-G5-G6-G7-G8-G9-G10
GelDoc 2018-08-27 T1-T2-T3-T4-T5-T6-T7-T8-T9-T10
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.
GelDoc 2018-08-27 16hr 13min H1 - H2 - H3 - H4 - Neg
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.
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.
-
Drylab Overview
20/8/2018
Great steps have been taken since last week, where we now have an approach to estimating the density of the mycelium.
Still trouble with 3d convergence.
21/8/2018
Try to create much simpler model to just try out the software, now have trouble visualizing the deformation.
24/8/2018
Used the knowledge and confidence from simpler model to locate, that the error was in the visualization. Successfully created a test video, of a combination of 6 bricks(see drylab folder), created a design with round edges to allow rotation