Moina as an Epigenetic model organism: CUBE Elphinstone

Moina as an Epigenetic model organism: CUBE Elphinstone
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No sir! I am not sure whether 2 Hb genes are expressed in normoxic conditions. It is just our (Mine, @Zahra_R17) hypothesis.

I should have not written that!

CUBE Elphinstone
Moina Group
@Zahra_R17 @saida786110

Objectives:
Immediate: Culturing of moinas and to maintain a red Moina culture.
Short term: To check for colour change in Moinas after a week or so.
Long term: To study epigenetics (the study of inheritable phenotype without altering the DNA sequence) and specifically DNA acetylation in Moinas using HDAC inhibitor Valproic acid (VPA) and to study the effect of unfavourable conditions like hypoxia in Moina and the probable reasons for the responses (changing to red colour) shown by Moinas in such conditions.

Today’s count:
L1 - 30-40 colourless moinas TE: 50 colourless moinas
Z3 - 20-30 colourless moinas TE: 40 colourless moinas
B2 - 10-20 colourless moinas TE: 30 colourless moinas
Z2 - 20-30 colourless moinas TE: 40 colourless moinas
B16 - 80-90 moinas with 30-40 pale yellow coloured moinas TE: 120 moinas with 30-40 red moinas
B1 - 40-50 colourless moinas TE: 60 colourless moinas
P1 - 70-80 with 10-20 pale yellow coloured moinas TE: 100 moinas with 10-20 red moinas
B3 - 100-120 moinas with 40-50 pale yellow coloured and 10-20 red moinas TE: 150 moinas with 20 red moinas
S2 - 10-20 colourless moinas TE: 30 colourless moinas
F1 - 150+ with 50-70 red moinas TE: 200 moinas with 70 red moinas
M1 - 80-90 colourless moinas TE: 100 colourless moinas
F6 - 40-50 colourless moinas TE: 60 colourless moinas
M6 - 40-50 colourless moinas TE: 60 colourless moinas

Further plan of work is to perform the VPA assay in CUBE HBCSE, Mumbai in the Diwali Vacation for which we are culturing red moinas.

And in the coming week we are going to prepare moina bottles at different milk concentrations.

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Moina group
Elphinstone college
@drishtantmkawale
Plan of work for 22/10/19
We have tried distilled water experiment 2 times but noticed that moina don’t survive in 1or 2 drop culture this is because distillation expels oxygen and hence moina dies but on Tuesday we will do aeration of water due to which distilled water will have oxygen.
Aeration water is done by bacterial shaker in which we can put the amount of water we want and aerate it.
What is aeration?
Water aeration is the process of increasing or maintaining the oxygen saturation of water in both natural and artificial environments.
The questions arose are:

  1. How & why does moina survive in 6 drop D/W culture?
  2. Does milk contain oxygen?
  3. After aeration of water how many days will moina survive in 1 drop culture?
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Today’s we (I, @Ameya @saida786110 @Zahra_R17 @mayur sir and Mansi) had a meeting with Dr. Subhojit Sen Sir from UM-DAE CEBS at Elphinstone college, we showed him our moina culture and we discussed about importance to various topics during experimenting and how we can improve experimental design by taking into account various parameters.

     In environmental science and particularly in chemistry and microbiology, a parameter is used to describe a discrete chemical or microbiological entity that can be assigned a value: commonly a concentration, but may also be a logical entity (present or absent), a statistical result such as a 95%ile value or in some cases a subiective value.

Reference : Parameter - Wikipedia

  In today's discussion, we discussed about variable and fixed parameters in moina culturing and how can we control these parameters or know their effects on the experimental setup so that we can formulate a better hypothesis. Also we discussed about how certain parameters are related to one another and what we can conclude from this e.g if the number of moinas (variable parameter) is high in a bottle, there will be increased moulting (also a variable parameter). 

     We further discussed on growth curve of moina which is similar to E.coli growth curve having a lag phase, log phase and stationary phase. How can we map the growth the growth kinetics in colourless and red moina culture bottles and comparison of these growth will help us better understanding of the growth of moina in normoxic and hypoxic conditions. 

      Keeping in mind the various parameters we also plan to design experiments to see how these parameters play a role in the growth of moina like : 
  1. Growth curve under normoxic and hypoxic conditions

  2. How does high concentration of milk play and role in redness on moina when the number of moina is kept constant

  3. Does the amount of dissolved oxyyen play a role in hypoxia if the moina culture is aerated?

  4. Will moina show hypoxia at a faster rate if they are densely populated in a small amount of water?

      While concluding the discussion we also discussed about the probable role of milk as a parameter and how better milk quality (pasteurized) can affect the growth of moina or slow the process of redness of moina and how can we formulate experiment to see the effect of milk quality and the use of milk powder as an alternative to milk...
    


After that discussion, the Moina group of CUBE Elphinstone highlighted some question mentioned above which we will figure out in the coming days.

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Yesterday, at CUBE HBCSE, Mumbai I and @Zahra_R17 from Moina model system talked about the Moina culture protocol, why is there a need for a protocol, components forming the cultures, the need for having a Control bottle too.


Photo credits: @Harshad Sir @Spandan_b

The logic behind the culturing of Moinas in Distilled water:

Usually, 250ml of DC Water + 6 drops of milk every alternate day + Colourless moinas → On the 7th day, ~30% pale yellow-red coloured moinas.

But, in our earlier Distilled water test tube cultures consisting of 10ml of DW water + 1:25 dilution of milk (1 drop of milk + 25 drops of DW) + Colourless moinas, we had made 8 such tubes (4 main + 4 duplicates). 1 drop of milk (control), 2 drops of milk, 4 drops of milk and 6 drops of milk dilution.
The moinas in the control tube had died when we observed on the next day.
The moinas in 2 drops of milk survived for 2 days.
The moinas in 4 drops of milk survived till the 8th day and were pale yellow on the 6-7th day.
Now, the moinas in 6 drops of milk test tube had become pale yellow to reddish on the 4th day itself given, that they were fed daily.

Our hypothesis was that they won’t survive in Distilled Water as its distillation expels gases, dissolved minerals and also the dissolved oxygen!!
So without oxygen and minerals, how will they survive!

But they survived and changed their colour too.
So @Zahra_R17 and I thought that if we are getting red moinas in Distilled water in a week, so why not try to have their Culture!

But, the question still remains, how are they able to survive in distilled water!?

So, to disprove our hypothesis, we decided to make culture bottles of moinas in Distilled Water.

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26/10/19
Cube HBCSE
@drishtantmkawale
Standard Protocol of Distilled water culture in 1000ml plastic bottles
Objective = To make moina culture in distilled water.
This method is for getting red moina way faster then getting them in dechlorinated water which takes approximately a week to become red
Procedure = we prepared 500 ml D/W + 20 (approx) moina + different concentrations of milk in 5 bottles :-
[ D/W 1 (Control 1) - 2 drops of milk,
D/W 3 - 6 drops of milk,
D/W 4 - 8 drops of milk,
D/W 5 - 10 drops of milk,
D/W 6 - 12 drops of milk ]
Expectations = our expectations is to check whether moina gets red in which concentration and at which day.
Questions arise are : 1) distillation deprives oxygen and due to adding more milk moina the oxygen gets deprived more so how do moina survive?

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@drishtantmkawale Can we understand from this regarding what your group think? Please clarify.

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What is the update on the effect of different amounts of milk?

knowing from @Ameya and @mayur sir that Moina macrocopa was found in CHM college the identification was done by ms. Kajal Kumari and that single line culture was made and is passed on to generations
that’s how we know that the culture we have is of Moina macrocopa only but we don’t know its distinct feature as for how it is Moina macrocopa only and not any daphnia or any other Moina

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CUBE Elphinstone
Moina Group
@Ameya@Zahra_R17@saida786110 @discobot

Research question: How the cells (of Moina in general) sense low oxygen and respond to the same by production of more Hb?

So, few weeks back…
@Arunan Sir⁩ had asked this question…

“What are HREs and what is its role in sensing the oxygen availability?”

Which was left unanswered by me…:grimacing:

So, HREs are Hypoxia Response Elements which are DNA segments that sense low oxygen availability and respond to the same by initiation of Transcriptional factors

There is a mechanism behind this which needs to be discussed here.

The question arises that how can a DNA segment sense low oxygen availability…
Or even sense something??

Reference: press-medicine2019.pdf (1.8 MB)

For HREs
https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(16)44683-6

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Hi! To find out what I can do, say @discobot display help.

@saida786110 are you and your group members clear now to distinguish between your Moina and the Genus called Daphnia?

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Text

How about doing an internet search?

Please post updates on Moina studies! Have you been able to establish a sure shot protocol which is at the same time simple to be done even at home?!! @drishtant @zaida

So, this is how we clean and change the water of the Moina Tanks at CUBE HBCSE, Mumbai

Procedure

  1. I took the Moina tank with 8L of DC Water in it along with the Moinas. Then I took a spare glass tank where I emptied the initial tank.
  2. A strainer or a sieve was used to strain the Moinas. The Moinas were in the strainer and the unclean water was there in the spare tank.
  3. I rinsed the initial tank with tap water and then took 8L DC Water in it.
  4. Then, the Moinas from the strainer were added in the tank by @KiranyadavR by swirling the strainer in the water so that almost all the Moinas are inside the water.

If we want to minimise the water usage, you can reuse the water unclean water.
How?
At CUBE HBCSE, we have Zebrafish and Guppy Fishes. These are filter feeders.
Filter feeders are animals which clean the water or environment by consuming the water, so we get clean water.
So, I had added 5 Zebrafish as well as Guppies to clean the water (I do not have a video. I forgot!:grimacing:)
Well, this was done for another tank.
Later, Kiran Ma’am added the Moinas into the clean water.

This is another way to clean the water.

This tank was made on 01 Jan 2020 and the water was changed on the 20th day i.e. 21 Jan 2020.

Why is there a need to change the water inside the Moina tanks?
As we add 32 drops of milk on alternate days, there is a huge bacterial load which would deplete the oxygen completely at some or the other point. There resides debris in the form of clumps which comprises of undigested milk, bacteria, rotifers, dead moinas, moults of the moinas.

This is not a standardised method to clean or maintain the tanks but it can be done easily without much hassle and the same can be followed with the bottle cultures too.

Video Credits: @Lydia

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Updates please! @drishtantmkawale and also list all collaborators in your college and also in other schools, colleges and locality labs!

26/01/20
CUBE ELPHINSTONE
MOINA GROUP
@Zahra_R17

•We prepared an assay of 7,8,9,10 drops of dilution (milk+water) on 16 January 2020.

•After 7 days we observed that Moina turn into red in only in 7 drop culture and in rest they died.

•This assay was to prove that why 6 drop culture is more suitable to use rather then 7,8,9,10 drops.

• Moina in 7 drops culture were less in number and hence we cannot take them in further assays

• 2 factors affecting Moina growth rate is turbidity due to more number of drops and less oxygen.
Hence proved that 6 drop culture is more suitable then 7,8,9,10 drops.

Major goof-up

We forgot to make the replicates and most importantly the Control test tubes!!

Replicates are important in the design of any experiment!
It happened so (in the CUBE X’MAS Workshop) that while looking for moinas in the test tube, it fell down and broke.
Fortunately, we had two more replicates of the same type.
In this experiment, we had only one replicate.

Why Control is important in the design?
We compare our test with the control.

It depends on the Objective of our experiment.
Here, our objective was to get Red Moinas.

So basically, we’ll experience a colour change and to show that the Moina has undergone colour change, we have a control consisting of 1 drop of milk/dilution fed to the Moinas so that their colour would remain the same till the test Moinas turn red or basically change their colour.

We basically use the Control to compare it with the Test ones.

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Research Question: Is Histone (remodelling) solely is responsible for the colour change of Moinas at low oxygen levels?

So if our Long term objective is To study the mechanism (histone remodelling) by which Moina attains Red colour at low oxygen levels this would make our Immediate objective as To get Red Moinas.

Why do we want Red Moinas?
Red Moinas would enable us to study their colour change from Colourless to Red and from Red, back to Colourless.
To study the colour change and how we can inhibit their colour change.
After getting Red Moinas after a week or so, we propose to treat the Moinas with different concentrations of Valproic Acid which is a Histone deacetylase (HDAC) inhibitor or in simple terms, Valproic Acid won’t allow the Red Moinas to turn back to Colourless.

By this, we will be able to prove that Histone Protein is inhibited from binding with DNA due to which the DNA would stay unbound and the Haemoglobin protein would be expressed in the form of Red Colour of the Moinas.

That would make our Short term objective as To perform the Valproic Acid experiment with Red Moinas.

Reference for Valproic Acid as an HDAC inhibitor:

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Cube Elphinstone
Moina group

@Drishtant @Zahra
Today’s report 1/2/2020
Today was the second day since we made the culture
CM - CONTROL
RM- TEST

M-MAIN
D- DUPLICATE

as we observe there is a slight colour change in RM
The moinas is started turning red already though they are juvenile

Number of Moinas observed to be increased in RMM and RMD

RMM - 20 -25 MOINAS
RMD - 20-25 MOINAS
CMM - 12-15 MOINAS
CMD - 12-15 MOINAS

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It’s a long message, spare some time for reading this although I’ve highlighted the important text!:grimacing:


Image credits: @Shivamkumar_Sriwas
So,
The day before yesterday’s Moina Group Causerie at CUBE HBCSE ended up bringing a change in everyone’s mindset!

We started with addressing @magpie issue of culturing Moinas in Kolkata.

Anyone reading this familiar with the culturing of Moinas may say that what’s a big deal in culturing Moinas?
Isn’t it?

If it wasn’t a big deal, we wouldn’t have had a couple of hours’ discussion on that!
Anyways,
We (@Arunan Sir, @yash_sheregare, @Shivamkumar_Sriwas and I) garnered the importance of having replicates in our design!

*Why are replicates needed?

  1. What if a :cat2: cat just knocks down the culture bottle?
    The setup would all be gone!
    We need to have replicates to make sure that if something happens to one or more bottles, we have others to rely on!
    Replica bottles must be the exact copies of each other!

  2. Having replicates also benefits us in checking the growth rate or dynamics of the Moinas!
    More the number of bottles, more number of data.*

For example, if we want to check the Moina growth curve, all the bottles containing the culture medium for Moinas, need to be made on the same day
So, as days pass by, we can check their increase in number, till when does it increase, when is the culture not increasing, and when does it starts decreasing and we can analyse the reasons behind such behaviour!

Not all the bottles would show the same result!
One bottles’ Moinas may start decreasing on the 16th day post-making and the other may start showing the same behaviour after 2-days i.e. on the 18th day maybe!

We need to understand the variable factors which affect the health of the bottle!
The factor which I pointed out which can be responsible (not solely) for decrease in the bottle was the increase of bacterial load, debris which may give rise to unwanted organisms like Rotifers.
But that does not make much difference!

Shyam Sir from CUBE Nattika was there in the lab yesterday.
He added that pH too can have an effect on the mortality of the Moinas.

@Arunan Sir added that ammonification/urea could also have toxic effects on them.

These are all the points to ponder upon!

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