CUBE Room Webinar!:- The Causerie will still be on!

CUBE Room Webinar!:- The Causerie will still be on!
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[5/11, 10:05 PM] Saida: in today’s discussion we talked about floral dip method in cardamine plant :herb:
floral :hibiscus:dip method is basically diping :droplet:the flower​:hibiscus: in plasmid and detergent.:soap:
plasma containing gene of interest
and we are using detergent :soap:so that it can make pore in the cell membrane of the plant so that the plasmid can get incorporated in it.
[5/11, 10:05 PM] Saida: and the gene of interest that we talked about was antibiotic-resistant Gene that is kanamycin resistant gene
so why antibiotic resistance gene?:face_with_monocle::face_with_monocle:
does antibiotic affect plants :face_with_raised_eyebrow:
certainly not antibiotic is used to kill bacterias and not eukaryotes😅
now the question is how would an antibiotic will effect a plant if yes then how🙄
[5/11, 10:05 PM] Saida: so as an example we took penicillin which affects the cell wall of the bacteria
and even plants have cell wall so can antibiotic effect plant cell wall
the cell wall of the plant is made up of cellulose basically glucose β(1→4)-glycosidic bonds.
and cell cell wall of the bacteria is made up of peptidoglycan. peptide is a compound consisting of two or more amino acids and glycans are compounds consisting of a large number of monosaccharides linked glycosidically.
so if penicillium affects the glycosidic bond of bacteria then and it can also affect plant cell wall. but Penicillin prevents peptidoglycan from cross-linking and cross linking is through peptide so penicillin will not be able to effect plant cell.
[5/11, 10:05 PM] Saida: now coming back to kanamycin. kanamycin prevents Protein synthesis and there is protein synthesis in plants so maybe it can stop
Protein synthesis in plants as well .
but kanamycin bind to the 30s subunit of bacterial ribosome. and in eukaryotes there is no 30s subunit of ribosome there is 40s and 60s so it should not affect plants but plants also have mitochondria and cytoplasm that are of bacterial origin which have 30s and 50s subunits of ribosome. so technically kanamycin can affect eukaryotes as well
[5/11, 10:05 PM] Saida: so supposingly if I take animal cells there is no cell wall as well only cell membrane and I take some detergent to interrupt the cell membrane of the cell and add some plasmid will there be a gene transfer taking place??:question::question:
[5/11, 10:05 PM] Saida: we have figured out how would kanamycin will affect the plant cell and if there is gene transfer of kanamycin resistant Gene then cardamine can be grown even in kanamycin.
but how would we by detergent disrupting the cell membrane of plant incorporate our gene of interest?!:face_with_monocle:

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18 May 2020, CUBE Room Webinar Summary

We started with @Rechel_tirkey ⁩ from CUBE Ranchi updating about her Moina cultures in her home lab.

Then, Gaurav Aggarwal⁩ studying at the Central University of Punjab joined us. We came to know that he has been working on Soil Worms or Nematodes.

We asked him about the things done by him before the lockdown.
So, as every nematode CUBist starts with, Gaurav also started collecting soil samples from the campus in a petri plate

Then, he prepared 2% Agar media and then poured it into another petri plate.

Why was 2% Agar used?

He also mentioned that 70% Ethanol was used by him as an attractant for the nematodes.
Why 70% Ethanol?
For me, it is used to make aseptic conditions and here it is being used as an attractant!
And Gaurav also said that Escherichia coli, a bacterium will be used by him to feed the nematodes.
I am sure that in their natural habitat, i.e. soil, not only E. coli, but many other bacteria would also be there on which the nematodes would feed.

So, Why specifically E. coli?
I suggested if bacteria are only needed, then the simplest source of bacteria would be milk which has nutrients in the form of carbohydrates, proteins, vitamins, minerals and fats as well as bacteria!
Instead of giving a Rajshaahi E. coli, milk bacteria can be used.

Can we start isolating the nematodes in our home labs?
What would be the procedure or design?

  • Originally asked by @Arunan
    @Anshu_kadam_2813 ⁩ @bivasnag⁩ @⁨Kunal Kadam⁩ @⁨Omkar Badnale⁩ @Shivamkumar_Sriwas - How will you guys start isolating the soil worms with the minimal required things available at our homes!!?

All this would be further explained by Gaurav.
This was done as his immediate objective was to isolate the nematode and identify it.

But @Lydia and many others questioned, how do we identify the nematode or the worm which we have got.
Gaurav⁩ said about DNA sequencing!
It is not a thing which we can do at home! Is it?

@PChitralekha added that sequencing can be done for identifying the animal or plant at the species level. She gave the example of Chlorella algae which at the species level, would need DNA Sequencing.

Arunan Sir⁩, @Lydia, Mayur Gaikwad Sir⁩, Aashutosh Mule Sir⁩, @yash_sheregare ⁩said that can’t we proceed in identifying the nematode taxonomically?
Caenorhaditis elegans is the soil nematode which has Nobel-Prize on its name!
So, we can start identifying the nematode and take the characteristics of C. elegans as the Control or Standard, to match with, along with pictures and references.

First of all, how do we know that the animal which we have is an animal?
If we start with addressing such basic questions, we will we able to get a context which we can relate to the school/college curriculum!

Taxonomy is anyways thought of as a boring thing to teach as well as to learn.
So why not make it interesting by discussing with the help of a whole library in our hand; our mobile phone.
Along with gathering some pictures and examples at each taxonomic hierarchy.

Answering questions like how can a soil nematode be differentiated from an Earthworm!? would give us a good start.
If we show a microscopic image of a nematode, a child would say, "Arey, this is a small Earthworm!"

If I show a layman, my Moina culture bottle, he would say, “Aye, ye toh keeda hai”! (Oii, this is an insect!)!
For them, it may be a "keeda* or an insect, but for us, we know that it is not. So, how do we convince people about our organism!!?

We can start by relating it to some other organisms which anyone could be familiar with!
If we see, Nematodes, Earthworms, Frutiflies, Moinas… at some point in their classification, they are grouped together say in Kingdom: Animalia, then as we go further, we come to know that there are some characteristics, which make them different from each other due to which some end up in the soil, some fly in the air, and some swim in the water!

The only wait is for getting a good context to start with, rest, the CUBists will handle!

We also discussed the taxonomical classification of the Standard Drosophila melanogaster CsBz fly and Moina macrocopa, both of which are Model-organisms in CUBE.

Later, in the end, @saswathy679 from CUBE SN College Nattika, Kerala shared her Fruitfly Homelab Medium story and updated us about the cultures and current happenings at her place.

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Online Discussions
17/05/2020 CUBE Room Webinar

We had discussions today on varied different
Topics starting from
Our special lockdown stimulated Home Made Medium for fruit flies - Tomato Rava SugarVineger Medium (TRS-V Medium)
To Moina Culturing, Circadian Rhythm studies in fruit flies, topics like Genetic drift in brief and many more!

Most highlighted Take home from Today’s Discussion

As @mayur sir added while discussing Moina Culture for Sweta CUBE Ranchi

Don’t put all your :egg: in one basket

In our CUBE , the context goes for every model system
Be it, Moina cultures (keeping of replicates)
Fruit fly cultures
Hydra cultures
Cardamine studies
And so on!
We need to have replicates for each time we try to do anything
Be it

  1. culturing or maintaining Moina cultures, or making a new setup for Subculturing!
  2. Maintaining fruit fly cultures and even testing home made medium for Culturing fruit flies!
  3. Culturing Hydra, Having replicates!

@⁨Ashwathy CUBE Nattika⁩ , a Nattika CUBist has very well tried and made a working home made TRS V medium which worked well to give rise to larvae (in three days) pupae (in another w days) and next generation fly (after another 3 days, which means next generation fruit flies in 8 days! ) in her single medium bottle! ( This result we see evidently also in our Std Corn Meal agar medium)

Now, to confirm and say, it’s a medium that functions well
Or lets say,
To make this our standard Medium, we all together need to Design and make medium with Replicates and control

Which means?
When we make and test the medium and say the medium is working well for getting us larvae, pupae and next generation flies, it shouldn’t be giving us the result in just one medium bottle but atleast 3 replicates and even better for more bottles tried and tested by all other CUBE centers working on fruit fly and culturing them during Lockdown Collaboratively!

For that we all together must discuss it in length with details
The design of medium
The protocol of medium
Modifications in the medium if any
Number of replicates to be made
Number of fruit flies added in each bottle (keeping in mind and also adding and counting number of gravid females in each bottle)
Types of Control setup needed?

Please add in your inputs! :smile:

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22 May 2020 CUBE Room Webinar

Yesterday’s discussion started with CUBist Abhijeet Singh, a Class XII student from Mumbai, sharing his experience in CUBE; he told us about the model organisms he worked on, the basic understanding of it. Abhijeet has worked on the Ventral Nerve Cord Regeneration in Earthoworms, on the Fruitflies as well and has also studied on Mosquitoes.
About Mosquitoes he told us about keeping Ovitraps. Abhijeet and @Hinaiqbal_Mudgal from Kanpur were discussing about their own experiences with mosquitoes, their current status and further plan.

Then, we came to the Drosophila-Fruitflies where @saswathy679 from Thrissur has been working a lot on the Fruitflies, be it trapping, medium preparation, preparing single line cultures. So, Abhijeet was new to this so we heard the summary of @saswathy679’s work so far and her further plan.

Then from South, we went to East-India in Asansol, West Bengal where @OYINDRIL is also working on fruitflies. As of now, the Circadian Rhythm Studies are being done by him.
We would like to know about your work @OYINDRIL DUTTA…!

@OYINDRIL is also planning to prepare media to maintain his fruitflies. So @saswathy679 is also working on fruitflies. As of now, the Circadian Rhythm Studies are being done by @OYINDRIL DUTTA…
We would like to know about your work @OYINDRIL DUTTA…!

He is also planning to prepare media to maintain his fruitflies. So Aswathy, who is now confident of sharing the Procedure (secret recipe🤭) of her media, we should look forward to get updates.
So her we saw that CUBE is acting as a connecting link between Thrissur, Kerala and Asansol, West Bengal where collaboration is taking place at every step and problems are being solved!

As @Arunan said, there is lack of anticipation/expectation from our work!
We should expect something in order to get something. If we aren’t expecting only, then what are we going to look for?
And also when proposing a hypothesis, design of an experiment, we should not do it vaguely but proceed with confidence so that the fellow viewers would be eager to know what is happening.
The way of presenting our work with proper headline/breaking news, plan, expectations matters the most!

Not to forget, Evidence based Research is what we should focus on, otherwise it would be like doing a ritual of which no one cares of!
Evidences, archives in the form of Research Papers (from recognized labs, universities, journals, authors/scientists along with cross references), Photos and Videos with a proper hypothesis/strong statement are the things which we should look for, further which would convince our audience!

For example, if we are preparing the Fruitfly medium at home, then we should start mentioning the quantity of media, the requirements of it, the texture, the procedure, cooking time…
If we say that 100mL medium was prepared just like that, then some fellow CUBist trying to reproduce the same medium won’t be able to do it.

Why?
Because we lacked in details!
This is why Arunan Sir always emphasises on giving the details because The DEVIL and the DEVINE is in the Detail!

But, if we go by saying that 100mL medium was measured using a cup or any other utensil, then it would be somewhat relatable otherwise people won’t do it saying that "we don’t have the sophisticated equipments for measuring and all… so we won’t proceed!"
@mayur added that simply relating the amount which we want by imagining/visualising the ~measurements which we use in our labs, will do the trick! (not necessary that the amount should be accurate everytime!)

Looking forward to get updates from everyone!

A short-summary after the Webinar will make sure that the discussion starts in the group!

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26th May & 27th May 2020
CUBE Room Webinar (A short summary)

While discussing in the webinar for the past 2 days,
@saswathy679 and many others were believing or claiming that temperature might be a factor that gave a delayed life cycle.

So, the story goes like this…:point_down:
@saswathy679 as a very good or a very thriving culture which started from 6th May.
Within 8 days, i.e. on the 8th day (14/05/2020), new generation flies ecloded from the pupae.
After 4 days i.e. on 18th of May, another set of media was made and the newly emerged as well as the parent flies from the 6th May bottle were transferred into the newly prepared 18th May media bottle.

In this 18th May TRSV media bottle, Aswathy could not locate any larvae for almost 7 days i.e. from 19th May to 25th May…but on 26th May, small and big larvae could be seen by @saswathy679 as a very good or a very thriving culture which started from 6th May.
Within 8 days, i.e. on the 8th day (14/05/2020), new generation flies ecloded from the pupae.
After 4 days i.e. on 18th of May, another set of media was made and the newly emerged as well as the parent flies from the 6th May bottle were transferred into the newly prepared 18th May media bottle.

In this 18th May TRSV media bottle, Aswathy could not locate any larvae for almost 7 days i.e. from 19th May to 25th May…but on 26th May, small and big larvae could be seen by @saswathy679 i.e. on the 8th day!

During the webinar,
Aswathy claimed that temperature might be the factor responsible for this delay.
When asked what was the temperature, it was told that it’s 27°C.

For me, and @yash_sheregare ,
We had 2 reasons for ruling out the factor of temperature!
:point_right: 27°C is normal (lab temperature) where we used to culture our fruit flies, the standard as well as the native single lines. And within 8 days we used to get the next generation flies.
:point_right: Aswathy herself got a thriving culture in the same place at the same location using the same media (without making any changes) few days back…not even a month old story…
And I looked for the temperature of those days.

There isn’t much drastic difference in the temperature that would affect the life cycle of fruit flies giving a delayed cycle.

If you ask me what was the temperature during those days.
I’m giving a reference along with the data of the temperature on all those days from 6th May to 16th May (the bottle which had a thriving culture) and from 17th May to 25th May (the newly made same type of media which was claimed to show a delayed life cycle of almost 7 days!!)

Reference: https://www.accuweather.com/en/in/thrissur/188812/may-weather/188812

May
6th - 34°/24° C
7th - 34°/22° C
8th - 32°/26° C
9th - 33°/25° C
10th - 33°/25° C
11th - 33°/25° C
12th - 33°/26° C
13th - 33°/26° C
14th - 34°/25° C
15th - 33°/26° C
16th - 32°/25° C

17th - 33°/26° C
18th - 29°/25° C
19th - 31°/25° C
20th - 33°/24° C
21st - 31°/28° C
22nd - 32°/25° C
23rd - 33°/29° C
24th - 33°/27° C
25th - 33°/25° C

@saswathy679 prepared media on 17th May too…
But, I emphasized more on 18th media as that was a replica of the media made on 6th May.
On 18th May, the 6th May media was replicated and flies from the 6th May bottle were transferred into these 18th May media bottles.
So we have the temperature range from 17th May to 24th May as well.

Do we really see a temperature difference or drastic deviation in these day??
I dont see any!!
Is it really a temperature bhagwan at fault or is it some other factors???

Today,
when we were again discussing about the same thing,
still we were stuck in the temperature thing!
Surprisingly, diapause & humidity factors also came up…okay…agreed…

So…What is Diapause?
A period of suspended development in an insect, other invertebrate, or mammal embryo, especially during unfavourable environmental conditions.
Unfavourable conditions!!!???
What unfavourable conditions did Aswathy or her flies faced??
Humidity was an answer…okay

So…What is Humidity?
Its the quantitative amount of water vapour present in atmosphere.
How can this affect the flies which are cultured inside a bottle which is kept closed all the time and is opened only when we have to transfer flies…??

Now, I would like to tell what could be the reasons along with the explanations for the so claimed delayed life cycle of Aswathy’s flies.

:point_right: According to @saswathy679’s observations,
She saw larvae on 26th May (both small and big)
And then, today i.e. on 27th May, she got pupae!!
Can a larva turn into a pupa within a day?
Its impossible according to my experience and observaions with the standard and the native fly cultures.
It takes minimum 2-3 days for a larva to turn into a pupa.

What can we infer from this?
Observation is lacking because strong expectation is missing
LOGICAL EXPECTATION…that has a great role in science, especially in DESIGN

I’ll explain it more…
If Aswathy saw a larva on 26th May and it turned to a pupa on 27th May…
That means there were larvae in the media 3 days before.

So…
On 23rd May, the flies had laid eggs,
On 24th May, the first stage of larva (1mm size)
25th May, second stage larva (2mm size)
26th May, third stage larva (3mm size)
On 27th May, i.e. today, we got to see pupae!!

So what happened??
Expectations were missing
Keen observation with interest was missing
Its ok, she might have missed out…this happens with many of us…but directly taking temperature as the sole reason is not right…
Human errors are to be first analysed.

Its not that temperature cannot affect.
But we need to have the proper data giving evidences that there was a drastic change in the temperature.
As well as we need to be sure that there wasnt any missing out of observations or expectations that happened from our own side.

:point_right: There could be one more reason,
A normal female fly can be mistaken for a gravid fly.
May be that those female flies weren’t gravid or fertile

This is my comment on the summary of the webinar that we had.
Waiting for comments from everyone.

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Summary- CUBE Room Webinar
Thursday, May 28th 2020

Moina-A brief discussion

The discussion started with the freshwater-crustacean Moina. I updated everyone about the Moina cultures at my home lab.
Then, I shared the breaking news of observing yellowish-green colour on the base and walls of the bottles.
I suspect that it is Algae which is taking over in the Yeast bottles. I added that once I see more greenish growth, I will add ~10 Moinas in both the bottles.
@KiranyadavR and Abhijeet Singh asked that how will the Moinas survive in those bottles? If we look at their natural environment which is ponds and lakes, the Moinas feed on Algae, Bacteria and Fungi (Yeast and others) present in the fresh-water bodies there.*

It will be interesting to know about their feeding mechanism on Algae and Fungi.

Abhijeet Singh asked about my Research Question and how am I working on that.
Research Question: How do the Moinas undergo colour change (colourless to coloured and vice versa) in less-oxygen condition or hypoxia and is it due to Histone modifications?

I said that it is very simple to induce hypoxia and observe the colour change of the Moinas. Everyone (if they have Moinas at their place) can do it in their home labs and the first possible colour change would be noticed by them in 72 hours!

And once the Moinas become coloured ones, we can turn them back into colourless Moina.
How!!?
By again keeping them back in Normal oxygen condition or Normoxia.
Here too, after a day i.e. ~24 hours or less than that, the 3rd day Pale Yellow or coloured Moinas will turn into colourless Moinas.

So we see that such simple experiments which can be done at home are the base of understanding the complex mechanisms like Histone acetylation/deacetylation, Remodeling and others.

Yesterday, a major goof-up happened by me that I burdened everyone present in the Webinar by explaining these complex mechanisms (without the help of any diagrams as such) instead of starting with the basics and explaining it simply!

My Apologies to everyone

I learnt that evidence based research is essential in explaining a complex system.
I have data, so I am able to explain.

Later, many others questions were asked.
Let us discuss them here. CUBists please repost the questions here.

Then, there came an issue of misunderstanding Milk as a Mutagen!!
@Vaibhavib99 and later @Shalu added that many people assume that the colour change of the Moinas from colourless to coloured ones and vice versa is due to mutations and reverse mutations respectively!

How is that even possible?
Does that mean many of us are consuming a mutagen?
No way!

Mutations take place when there is a change in base-pair sequence of the DNA.
They take time to get expressed.

And as I mentioned earlier, only the configuration of DNA and Histone is changed and rest all remains the same.
THE DNA SEQUENCE DOES NOT CHANGE!

So milk in any form, is not a mutagen which will cause mutations in Moinas!!
This is where we go wrong in understanding the TEXTBOOKISH things!

Instead of ending it here, we took it somewhere else.:grimacing::man_facepalming:

Fruitflies-the flies on demand

We discussed about the current status of the fruitfly cultures maintained by @saswathy679 at her home lab in Thrissur, Kerala and @Hinaiqbal_Mudgal too, at her home lab in Kanpur, Uttar Pradesh.

As they are having many bottles with as many flies in them, all of us suggested that they should start with the identification of the dead frutiflies in their bottles.
Even if they do not belong to a single line culture of flies, any random fly can be used just as a warm-up for identification.

Starting with the family identification and moving further on.
Yash, Aswathy , @Lydia and others please add to this and let us start with the identification procedure and its design!

Shedding some light on the slow-mo guys: Snails
As we were wrapping up, we came upon the Snails which are maintained by @Anjani from CUBE Elphinstone, Mumbai, in his home lab.
His collaborators Manasi Prasad, Nishmita Amin, Nikita Dhuri updated that one of his snails died.
How do we know that a Snail has died? It may be that the snail must be inactive or sleepy!

They said that a stench was coming from that snail.
Was that a result of any bacterial, fungal infection?

Zahra Risalawala said that she had read a paper (please share that!) where it was saying that the excess of salt can harm the Snails.
Is it?
What will happen if we take a mouthful of salt?
What happens when our fellow Earthworm is exposed to high salinity.
I have seen that the earthworm tries to move away from it.
It may shrink too.

Why?
Does dehydration take place?

​Nishmita Amin said that it is osmosis taking place.
What is osmosis? Do we really understand it?

We can simply start saying that because there is high salt concentration outside and low/normal salt concentration inside the body cells of the Earthworm, the salt will try to diffuse inside and in the process, it will dehydrate the Earthworm as more salt would be present inside which is harmful.

Why is it harmful?
Is it not a CONTEXT TO CURRICULUM question?

Let us solve it!

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Which curricular questions are being addressed here? @drishtantmkawale Please like at them with an one line description.

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Nematodes are generally found in soil , garbages, etc. So following the procedure of placing the soil sample on the periphery of 2% Agar surface and then adding the feed at the centre and thus isolating the nematodes is possible. But during the Lockdown some requirements such as :-

  1. Dissecting Microscope.
  2. Agar .
  3. Petri Plates ,etc. Are not present at home. So the isolation and identification of the nematodes might be a problem. Even the foldscope would just show an organism like a nematode but it can’t be proved as the Characteristic features won’t be visible through it.
    I guess isolation or even culturing nematodes during a lockdown would be difficult at Home labs without the instruments or material mentioned above.
    I am still searching for alternatives that can be used to observe the nematodes and a culture/surface media that can be made at home instead of using agar . Will come up with some idea ! Others can also look for some ideas that can ensure the idea of culturing and identifying the Nematodes at our home labs. This would come up with a great result and create new alternatives for the species to work upon from Home Labs.
    @Anshu_kadam_2813 @Arunan @bivasnag @Bunny @Shivamkumar_Sriwas @Ritam007 @Garima_kalakoti @jaikishan @drishtantmkawale
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I would like to contradict you @Kunal_Kadam.
You must be familiar with the current happenings in the CUBE Fruitfly Model Organism group.
We are doing fantastic work there. A home-made fruitfly medium has been devised (incomparison with the Standard Cornmeal Medium) to culture fruitflies at home and it is working incredibly well!

Why not we think of doing the same here?
I bet no one in the world must be doing things at home which CUBists do!!

Not even isolate, but can’t we just look for nematodes or even soil worms.
Dissecting Microscope is too sophisticated! Our phone camera shall do the trick.

Well, we can find alternatives for Agar! As the lockdown is easing now, we can make a search for agar alternatives and then start with it!
Agar, if we see is used as a solidifying agent or gelling agent (the lab one’s are too expensive!).

As mentioned earlier, the Fruitfly guys have found out Rava/Semolina/Suji as an alternative for agar.

Petri plates can be replaced by transparent lids or plates maybe.

Pinning Prof. Obaid Siddiqi’s Quote

"Sophistication should be in mind, not in lab"

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@drishtantmkawale
Here you are comparing two Different Model Organisms !! They are different in Size , Characteristic and even feed and substrate . Keeping in mind the size of the single Adult Nematode is 1mm approx. The mobile phone cannot zoom in to that level as it does help in case of the drosophila as it is visible through the naked eyes too… Now talking about the isolation of the nematodes - the nematodes are found in soil , it is very tidious to islolate them from the soil.
The media on which we would place the soil should not get affected by the soil itself. And the media should not get affected by the bacterial feed too . If we compare the transferring of the nematodes from one media to another would be more tidious work to do… As in drosophila the bottle transfer is done by the drosophila otself by flying to the other Bottle.
Soo it’ll be little difficult to find an alternative but not impossible !!! If everyone put forth their views or thoughts on this !!! It’ll be easy , not that difficult to find alternative for all and start with nematode isolation at home!!!

@Anshu_kadam_2813 @Bunny @bivasnag @Arunan @jaikishan@ @Ritam007 @Garima_kalakoti

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