Sentient cell experiment --> Get cells from baby goat, culture cells to multiply them, infect cells with virus containing my proteins, amplify virus, make cells produce the proteins, harvest and kill cells, purify proteins, test protein cocktails on mice
Table of Contents
Notes for lab assistants:
- Need to find optimal concentration for cells or will not grow properly.
- Cells continuously multiplying --> need to feed them new culture medium every 2-3 days or will starve + dilute cells once or twice a week with new medium to give them room to grow or will die
- Keep sterile protocol to avoid contaminations!!!
- When I say check every hour, I mean every hour of the night too!!!!! I need precise measurements!!!
- If mess-up experiment, Dark Lord will deal with you!
Cell harvesting - Day 0
In farm: Harvest cells from baby goat H7 after birth.
- Make cuts per protocol and sample cells.
- Put in glass tubes and keep on ice.
In lab: Start sterile protocol + start cell culture by putting cells in 12-well plate.
- Feed the cells 1 mL DMEM-LT culture medium instead of simple DMEM like last time --> may take longer for them to get use to it and thrive but should make them healthier.
- Put in incubator, 37°C, 250 rpmON.
Check on cells - Day 1
Still alive, unhappy. Form small clusters instead of being properly diluted in medium. Probably want more friends around again!
--> Don't transfer cells now, wait longer to see if multiply enough to get to viable conc.
Cell check - Day 3
Cells even more unhappy and still not settling. --> Centrifuge 5k rpm 5min.
- Get rid of medium and resuspend cell pellet in new 1 mL medium.
Cells check - Day 5
Damn cells died on me again! They're doing it out of spite!
--> Schedule new harvesting. Bully Dark Lord to get him to find new pregnant goat soon!
New baby goat born during the night. Harvest cells H14 --> late but might help them survive better.
Use same plate as last time but only use 500 uL medium to get them more concentrated --> stupid cells always feel better with friends.
Cell checkup - Day 5
Contamination!!! Cells swallowed bacteria on purpose to protect bacteria from antibiotic and suicide themselves!!!
--> Get Dark Lord again and redo everything with new goat + avoid letting cells see rest of lab and what happens to other cells so that don't try to avoid their fate!
Another contamination!!! Damn new lab assistant not following proper sterile protocol! And contaminated back-up flask too! Need to restart everything!
Cells doing ok finally! Will be able to start proper experiment next week.
Dilute cells a bit more to make them last longer --> No way I'm working over the holidays, Dark Lord or no Dark Lord!
Cells not happy about dilution -_-
Centrifuge 5k rpm 5min to get read of medium and reconcentrate the cells. Delay experiment by one week until cells recover.
Cell culture by AmélieIS
--> cells need friends nearby to make them feel better or will pout and kill themselves out of spite!!!
Cells ok + concentration great! --> infect them with the virus, same conc as last time
Observe cells with visualisation gnTL
6/12, Day 0 = no colour
7/12, Day 1 = no colour (cells unhappy, but can't do anything while fighting to survive the virus!)
8/12, Day 2 = start to see colour
9/12, Day 3 = max blue and green colour --> centrifuge cells, collect medium to keep the virus and throw away cells right in the bin to give them time to suffer as they die off 10/12
Using uninfected 1 L batch-2 of cells, infect them with 10 mL virus to amplify it.
--> cell batch 1&3 standing next to batch 2 see it be infected & really unhappy. Start buzzing for 1.5h before exhausting themselves = good time --> confirm that healthy cells.
Use all 3 batches of cells left and infect with amplified virus, use 30 mL virus for 1L cells.
Cell check up
Cells all died off!!!
-->Damn lab assistants kept poking cells to make them buzz, exhausted them and now too tired to fight off virus
Need to restart cell culture to get big concentration to redo proper infection = will take 3 weeks!!! Get assistant to work over Christmas to teach them!!!
Cell check up
Lab assistant-3 thinks he's very clever. Keep messing up with the cells. Want to teach them tricks & make them react to his actions in lab --> not going to end well. Give him one cell batch and set him in lab 5 away from my Very Serious Work!
Lab 5 check up
- Lab assistant-3 disappeared, another traitor -_-
- Cells from lab 5 all dead or asbent (too diluted?).
- Glassware not cleaned!!!
From lab 5:
Suspisious traces by AmélieIS
Cell check up
Cell doing ok after infection. Colour check up is ok --> can harvest them tomorrow to get max amount of protein
Harvest cell + prepare purif
Harvest cells from flasks by centrifugation. 5k rpm 5min.
- Get rid of medium, resuspend in binding buffer (5mM imidazole, 50mM Tris, 150 mM NaCl) in 12 different flasks.
- Sonicate each flask 10 sec on 10 sec off (--> time off avoid them heating too much and burning) for 10 min.
--> Put other flasks next to sonicator to show cells what awaits them! That will teach them to be so annoying! - Prepare purification: wash machine + equilibrate purif column (use column type AB2) with 5 CV binding buffer = 5x5 mL = 25 mL.
- Load cell lysate on column.
- Do 10 CV of wash buffer (20mM imidazole, 50mM Tris, 150 mM NaCl) = 50 mL.
- Do gradient 5 CV for every 20% increase of wash buffer vs elution buffer (500mM imidazole, 50mM Tris, 150 mM NaCl).
- Collect proteins from first UV peak.
Sonication by AmélieIS
Make cells burst open, collect resulting cell lysate.
Purif strategy - Column will bind the protein I want but not all the other proteins produced by the cells.
- Load cell lysate on column --> most impure proteins don't bind
- Wash column with wash buffer --> proteins that bind non-specifically get taken away from column.
- Make gradient with wash and elution buffers go through column --> my proteins are first to detach and be eluted.
- At higher gradients, it's impure proteins that detach. Do that to clean the column for next purif.
Test different proteins cocktail
Need 1-1.5 years old mice --> use 14 months old, 2 mice per protein mix --> one mouse try to reattach tail, one mouse try to get full tail regeneration
Get lab assistants to check every hour this time! Get Dark Lord to tell them next cells will be harvested from them otherwise!
1) 50 uM GhD1+100uM LDFnd1+100uM LpNd
2) 50 uM GhD1+100uM LDFnd2+100uM LpNd
3) 50 uM GhD1+100uM LDFnd3+100uM LpNd
Time 0 = Cut tails off.
Time 5 min = Inject one protein mix in back below the head.
Time 10 min = poke mice to cause emotional distress to get them to metabolise proteins.
1) Mouse 1: H13 tail fully reattach
Mouse 2: H24 tail grown back by 1.3cm
2) Mouse 1: tail not reattached at H72
Mouse 2: H24 tail grown back by 0.2cm
3) Mouse 1: H21 tail fully reattach
Mouse 2: H24 tail grown back by 1cm
--> Cocktail 3 mouse 1 = should have been earlier than H21, damn lab assistants again too lazy to check every hour!!!
--> Harass Dark Lord until do something about it!!!
Meeting with Dark Lord
Dark Lord complains about emotional distress on mice because won't be able to do it on future customers. All wimps! -_- Could spare some tears for the most revolutionary medical regeneration process ever!
Further optimisation in mice useless anyway. Mice brain too different from humans, will affect metabolisation of protein cocktails --> need human test subjects!
Meeting with Dark Lord
He got me lab assistants as test subjects! How am I supposed to keep prisoners in the lab? Will make a mess and destroy sterilisation! He better gives me a proper jail extension, I don't care about the cost!
And a new lab assistant, I can't be expected to do without! And while we're at it, lab 5 needs full renovation.
Will bully him until get it
Want to learn more about sentient cells? Check the textbook!