Team:Alberta/Methodology/Notebook/Growth

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METHODOLOGY

Notebook

We procured a strain of Neurospora from a neighbouring lab and set out to learn how the organism grows. We made potato dextrose media and tested the growth of Neurospora in the presence and absence of glucose.
We also tested out household fertilizers as a replacement to the potato component in varying concentrations, we did not observe much growth in just fertilizer and glucose. We examined the fertilizer and found that it was missing Magnesium and Calcium. We created a salt solution and measured growth on fertilizer and glucose with our added salt solution.
We created primary experiments on saw dust and coffee grounds.

Tested the growth of Neurospora on strained versus unstrained potato dextrose media in varying concentrations.
We set up a growth curve on potato dextrose media in 100mL volumes to attempt to create a standard curve.
We set up small scale experiments of sawdust and coffee grounds with promising results.
We made a variety of media from grass clippings and wheat straw. We tested chopped versus ground components of each and also tested NaOH pre-treatments of wheat straw.

We learned about how to properly inoculate with conidia and correct procedure for growing and harvesting conidia.
We attempted a scale – up of the successful small scale Wheat straw and grass clippings experiments from last week.
We created media from phone book and baked sawdust and preformed a small scale experiment.
We tested varying amounts of distilled water in grass clippings and saw dust.
We attended the AITF workshop in Calgary.

We miniprepped pMOCosX G6C9 for the growth team. We transformed pCSN44, and pBARKS in to DH5alpha cells and plated on Amp agar plates.
We attempted to perform a small scale growth curve in potato dextrose media.
We tried to make petri dish cultures with liquid media of wheat straw, grass and potato dextrose.
We made VSuTB (no agar) and tried to make a small scale growth curve.

We decided to narrow our scope and focus on perfecting the growth of Neurospora on grass clippings and wheat straw. We felt that grass clippings represented the household application and wheat straw represented a large-scale/ industrial application.
We created a wheat straw experiment varying the size of wheat straw added and in a separate experiment, the amount of water added to wheat straw. We also compared additives.
We created a grass experiment comparing dried grass to freshly cut grass.
We made our first race tube out of a 25mL disposable pipette.

We tried to make another growth curve from Vogel`s minimal media.
We made a wheat straw scale up in 100mL from the most successful results from the previous week.
We set up a dried grass small scale experiment varying size, water, and additives.
We inoculated our “race tube”
Kayla attended the ISMOS-3 conference in Calgary.

We miniprepped pCSN44 for the growth team.
We acquired long glass tubes to act as our race tubes.
We filled them with VSuTB and inoculated them with fresh conidia. We marked growth along the tube every 24 hours.
We made tubes of Wheat Straw and VTB and tubes of Grass Clippings and VTB. We inoculated the tubes and monitored growth of the tubes every 24 hours. We also noticed that it was difficult to see growth in the clear VSuTB so we made two tubes of VSuTB, one with food colouring in it and one without. We tested growth and found the hyphal extension rates to be comparable.

We attempted to make a scale up using wheat straw. We continued to monitor and record growth if our race tubes.
Week 9 July 4 – 10
We set up new race tubes to test if the carbon source being used was actually from the wheat straw and grass clipping. We made the following tubes up: VSuTB, wheat straw +VTB, wheat straw + fertilizer + salt solution, grass +VTB, grass + fertilizer + salt solution.
We also attempted to extract the Neurospora from the race tube by boiling the agar out but this had very limited success.

We ran a ApaL1 digest of TesA’ gene construct in pUCminusMCS for the genetics team.
We began testing parameters and setting up the camera and program for our time lapse video trial run.
We made 9 different media for each of our 9 race tubes for the time lapse video and harvested and counted fresh conidia to inoculate the race tubes.
We began our time lapse trial run.
We made growth based slides and compiled the team’s slides for our lab presentation.

We diluted primers received from IDT for the genetics team.
We made a presentation to show at the AITF workshop
We attended the AITF workshop in Calgary.
We received three GFP plasmids, pMF 272, pMF 280, and pMF 309, from the fungal genetic stock center. We prepared these plasmids for transformation and transformed DH5alpha cells with the three plasmids. We also prepared mini-preps of the DNA.
We set up 2 PCR reactions of the Hyg B gene from pCSN44 for the genetics team.
We ran a magnesium gradient PCR of the TesA’ gene for the genetics team.
Tested grass clippings and wheat straw in 100mL cultures.
We lived the dream.

We made potato dextrose media. We split this media into 100mL portions in baffled flasks. We inoculated these flasks with fresh conidia to grow enough Neurospora for the production of biofuel to test in a diesel engine. The Neurospora was incubated for 72 hours and dried before extraction and esterification.
We set up materials and prepped for the real deal time lapse video.

We made 9 different media for the time lapse video. These were autoclaved, poured into the race tubes and allowed to set.
We set up the camera and computer with our specifications discovered from the trial run.
We inoculated the race tubes and began our time lapse video.
We started a growth experiment to determine the amount of Neurospora produced on liquid cultures of wheat straw and grass clippings and VSuTB.

We transformed FatB1 into DH5alpha for the genetics team. We miniprepped and plated FatB1 on Amp plates.
We continued our liquid culture experiments but encountered some difficult with the Neurospora continuing to grow after removal from the liquid medium.
After much research, we met with a local Edmontonian model plane expert to discuss diesel engines and buying a plane.

We worked on accumulating Neurospora cell mass to esterify to power our diesel powered engine. This was done in 1.5L potato dextrose media, grown at 30C at 175rpm for 72hrs.
We worked on a video to described our project to the public and prepped the project abstract to submit to iGEM.
Set up a race tube experiment. This experiment comprised of 3 grass clippings +fertilizer agar tubes of 100mL each, 3 wheat straw + fertilizer agar tubes of 100mL each, and 1 VSuTB 100mL tube to act asa control. We counted fresh conidia and inoculated the race rubes with approximately 15 000 conidia each. Growth of Neurospora in the tubes was monitored for a week.
We grew another mass of Neurospora in 1.5L of potato dextrose media.

We graphed our data and assembled the data and text needed for the wiki and presentation.