Success: rsh Stock has rsh1 Mutation

on Monday, August 7th, 2023 11:11 | by

Via gDNA analysis and PCR was the specific area of the rsh gene extracted and amplified where the nucleotide substitution: C to T (Folkers et al., 2006) should be for the rsh1 mutation. The amplicon was Sanger sequenced which proved the nucleotide substitution.

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Bachelor Blog / #3 finally, data!

on Monday, July 24th, 2023 2:22 | by

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Little by little

on Monday, July 24th, 2023 1:59 | by

Despite very warm weather, some flies did fly, even though the learning performance of the control flies was really poor. At least for now, it looks like all stocks are learning and that rut and rsh flies learn at least equally well as the Berlin flies. I’ve also managed to fix the positive preference problem:

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Creating gRNAs via PCR

on Friday, July 7th, 2023 6:38 | by

1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 640 pg/µl.
1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 64 pg/µl (1:10 dilution).
1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 64 pg/µl (1:10 dilution).
50µl of 5xQ5 High GC Enhancer was added to the PCR mix.
1% Agarose gel with 100bp marker and PCR1 rsh, PCR1 rut.
The template pCFD6 was used with a concentration of 128 pg/µl (1:5 dilution).
50µl of 5xQ5 High GC Enhancer was added to the PCR mix.
1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 640 pg/µl.
The Phusion DNA Polymerase was used instead of the Q5 High-Fidelity DNA Polymerase.
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Kicked out Canton S

on Thursday, June 29th, 2023 5:25 | by

Since the Canton S strain I used wasn’t a perfect genetic background strain anyway and didn’t fly properly, I kicked it out and replaced it with the wild type Berlin data I had collected just prior to the rut and rsh mutants. Now I can collect data for two projects in one go: I check the rut/rsh learning mutants if they still behave the way they should and with the wtb control strain and continue collecting data to evaluate their optomotor responses after training. So now the learning scores for the three strains look like this:

It still doesn’t look like rut is better than wtb, so I’m still skeptical that the strain is really what it should be. The rsh data also don’t look very promising, but I still need to get more flies with a negative preference before the training.

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Suspicion and progress!

on Monday, June 26th, 2023 2:03 | by

Suspicion: rut may not be rut

Carsten Duch is identifying the muscles involved in operant self-learning:

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It’s a start

on Friday, June 16th, 2023 4:42 | by

The first few rutabaga and radish mutants have been measured and so far it looks good: standard duration yaw torque learning – the situation all three groups should learn.

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Bachelor Blog / #2 problems, problems

on Monday, June 5th, 2023 12:59 | by

Hello everyone!

Things are not going well…

Over the last week I have encountered numerous problems in carrying out the planned experiment, and the biggest of these is definitely the generation of offspring. Since it has been shown that long-term memory decays quickly in the first 24 hours after operant self-learning on the flight simulator (and with that possible epigenetic marker I suppose), I decided to mate the flies immediately after training and separate them again after 16-20 hours. However, I did not get any offspring by doing this. I tried mating virgin males/virgin females and also adult males/virgin females for 16-20 h, but no eggs could be found in the vials. I have not yet found out what prevents the flies from mating and am now trying again with older (3-4 days old) adult male flies. 

Another problem is the training protocol for memory consolidation by S Xia, L Liu, C Feng et al. 1997. Although I have practiced gluing and inserting flies into the flight simulator, I don’t think I can train enough flies in one day. To maintain sufficient larval density after mating, I need at least 5 females and 2-3 males, and I would need to train them twice, giving a total of 16-18 training rounds per day (and that doesn’t include failed attempts…). Alternatively, I can use the standard protocol and see if the training is enough to reveal epigenetic inheritance of acquired habits, if any. 

For now, I will continue to try to produce offspring and practice gluing and training the flies. I have started to do further research on inheritance mechanisms to see if it might also be possible to work with only the F0 generation and detect epigenetic markers (methylation of genes, e.g. foxP, siRNAs) in the trained flies and compare them with untrained flies. Here (1, 2) you can find a few interesting articles on this topic.  

However there is also some good news to report: my ContonS line will be ready soon, so I don’t have to work with the wtb virgin flies anymore, which decease very quickly. I have also made some progress on the statistical background after talking to a friend of mine although I still need to do some research on how to set my effect size so I can finally calculate the sample size I need for the experiment.

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Bachelor Blog / #1 outline

on Monday, May 22nd, 2023 8:45 | by

Hello everyone!

Here is a brief overview of the topics I will be dealing with over the next few months:

This is the experiment by MD Ziv Williams I am trying to imitate ->

If you are further interested in transgenerational inheritance of behavioral traits and non synaptic memory storage check out these interesting links ->

This week, for now, I will continue practicing to glue the flies and make a realistic estimate of the number of flies I need to get a significant result. Hopefully I can start trying out my experimental setup by the start of next week :)

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Machine works, flies are not learning

on Monday, May 22nd, 2023 11:48 | by

The old software is installed with the new hardware and I’ll be testing the new software in parallel to experiments with the old software. Technically, everything seems to be working now, but the first six flies I tested did not show learning after training:

However, avoidance looks fairly ok, only the last training period gives away that something still isn’t quite the way it should be:

So I’ll go ahead and train a new batch of flies this week. I’ll focus on training/avoidance and make sure it is perfect, before I shorten the training.

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