amino labs Extract it Kit Extract Biopigments from Bacteria User Manual
- June 5, 2024
- amino labs
Table of Contents
PLATE EXTRACT-IT KIT™
USER MANUAL2018-08-23
www.amino.bio
Version 3.3, Oct 2023
©2015-23, www.amino.bio, Amino Labs
**Welcome! Let’s get started
**
This User Guide was created to help you get the most out of your Amino Labs
Experience. Even if you are familiar with genetic engineering, science or
other Amino Labs™ products, please take the necessary time to read through
this guide. This will ensure you practice safe science, store, use and get the
most out of your Kit and importantly, know what to do in case of a spill or
accident.
In the first section, you will learn about your kit’s components, how to store
them before and during your experiment, as well as a few tips on activities to
complete before you get your hands wet. The second section is procedural —
these are the step by step instructions on how to run your experiment. Make
sure to follow our tips to ensure your best success! The third section covers
“what’s next”; how to keep your creations, store or dispose of any leftover
ingredients and general clean up instructions. The final section is there to
help you — a glossary, troubleshooting, and our contact information.
Amino Labs is excited to welcome you to the world of the Genetic Engineering
with the Engineer-it Kit™, Canvas Kit™, Extract-it Kit™ and our entire
ecosystem of easy-to-use, easy-to-succeed at products!
Following this guide will help ensure that you are getting the most out of
your current and future experiences to keep on making new creations with DNA.
Have fun!
Practicing Safe Science
Genetic engineering and life sciences are safe activities when you follow
simple guidelines. Read on to ensure you adopt safe practices.
The kit in your hands contains only non-pathogenic ingredients. These are part
of the biosafety Risk Group 1 (RG1) (Biosafety Level 1). This is the most
benign level and therefore the safest: with these kits, no special containment
or training is required in North America. But you must follow these safety
guidelines for your safety and the success of your experiment(s)!
We recommend the system and kits for ages 12+, under adult supervision, and
14+ with or without supervision. We recommend that an adult empties the
discard container. The cleaning instructions must be strictly followed for
safety and experiment success. Make sure to store the kit per the instructions
found in this booklet.
- Do not eat or drink near your experiments. Keep your experiment at least 10 feet from food, drinks, etc. Under no circumstances should you eat any of the kit’s content.
- Immunocompromised persons: While the ingredients in these kits are non-pathogenic, some persons, such as immunocompromised persons, can be affected by large numbers of bacteria and should talk to their doctor before doing any experiment.
- Wash your hands before and after manipulating your experiment, or the hardware.
- Wear gloves, even when cleaning your station or handling the kit contents (petri plates, loops, etc). This will protect you from your experiment, and your experiment from you. Any latex, nitrile, or general purpose gloves you can find at the pharmacy will do. After you put your gloves on, be aware of what you touch. Try not to touch your face, scratch itches with your gloved fingers!
- If using the DNA PlaygroundTM or BioExplorerTM place it on a stable work surface. Keep it level at all times.
- Clean up your station, spills and work surface before and after use. Use a 10% solution of chlorinated bleach generously sprayed onto a paper towel and rub onto any contaminated surfaces. (Careful! This can discolor your clothes). A chlorinated spray cleaner also works.
- Find a container to hold the inactivation bag where you will discard used consumables. An old 1L yogurt con- tainer, large plastic cup or the like will do. Used consumables will be loops, any tube or used petri dish.
- Eye-wear is not provided but can be worn.
You can download a biosafety poster for your space from
www.amino.bio/biosafetyinaction
and complete a short safety quiz at www.amino.bio/biosafety-
quiz
If you would like to do a short Online lab safety course for your edification,
we recommend a Government of Canada course:
www.amino.bio/biosafety
Discover your Plate Extract-it Kit™
For an end-to-end bioengineering experience, Amino Labs provides you with the
means to extract and purify what your bacteria produced so you can use it
outside of the system. In other words, The Extract-it kit allows you to take
the product created by your DNA program plasmid (for example, a coloured
protein) from within the bacteria so that you can use it.
First, the bacteria’s cell wall will be broken open, and then filtered out so
that you can obtain a solution of proteins. You will then filter this liquid
for sterilization. What is cool about this is that the DNA program is still
present within the product, so that if someone ever wanted to, they could copy
it from there, and grow it once more in bacteria!
Specifically, your kit will allow you to complete the following hands-on
exercises to successfully achieve protein extraction :
- Grow more bacteria to “amplify” the amount of protein you can extract.
- Collect bacteria and centrifuge it down into a “pellet”.
- Lyse (break open) the bacteria using surfactant and enzymes.
- Collect and filter the proteins.
Individual kit size
The Individual kit size will lets you complete the experiment in full, one
time! This kit can be used alone, (with parent supervision if necessary) or in
a small group.
Group kit size
The Group kit size contains 4 individually-wrapped student packs and one
shared materials bag that contains blank cells, positive control cells, DNA
program, and the inactivation bags. These items will be shared by the group.
In the students packs, you will have everything else you need for the
experiment to be done 4 times by student groups or individuals.
Kit Components
Inside the Extract-it Kit™, you will find these components:
| Lysis buffer: : softly breaks open (lyses) the cells to release the cell
contents. This buffer should be used in concert with Lysis Accelerator.
---|---
| Lysis Accelerator: includes enzymes that break down the cell wall of
bacteria and works with Lysis Buffer to release the contents of cells into
their environment.
| 0.22 um filter : This filter has pore sizes that are 0.22 um which are
smaller than bacteria. This means bacteria cannot pass through, but your
pigment (smaller than 0.22 um) can.
| Syringe: Used to push unfiltered extract through a filter. Caution! Do not
press to hard to avoid liquid mishaps. Goggles recommended when using the
syringe.
| (2)1.5 mL Screw Cap Tube : Use one to store your final, extracted and
filtered product, and one to use as a balancing tube when microcentrifuging.
| Burst bag: a plastic bag to use over the syringe-filter sterilization to
minimize possible mess.
| Pipets : Pipets are used to transfer the cells between tubes.
| Agar, Sterile Water, Plates, Antibiotics, Loops : Just like the Engineer-it
Kit, these components are used to make selective LB agar. The loops are used
to streaked your already engineered cells as well as to harvest your grown
engineered cells into Lysis Buffer.
Unpacking and storing your kit
For a better shelf life and successful experiments:
- place your Extract-it Kit™ bag in a standard refrigerator at around 4°C.
- place the smaller Freezer bag with the brown tube(s) in a freezer.
Do Not Freeze all of your kit!
Do not leave tubes at room temperature!
Necessary equipment
For Best results:
- DNA Playground™ or BioExplorer™
- Microwave
- Microcentrifuge
Alternative solution:
-
Microwave
-
Microcentrifuge
-
Thermometer (for 42°C)
-
Timer
-
Ice bucket or bowl and ice: This will become your “Cold station” “Ice” for the experiment. Make sure to keep the ice from melting too much during the experiment.
You may need a fresh replacement during the experiment if it is warm where you are. -
Hot water bath or bowl with hot water: This will become your Hot station set to “Shock/42” for the experiment. Heat the water to 42°C and try to keep it as stable as possible while you heatshock.
-
Incubator or warm environment!: This will replace the Incubator set to “37”. This will replace the Incubator set to “37”. If you do not have an incubator (biology or egg one, as long as they set to 37°C), you can create one using an online tutorial Search for DIY incubator on our youtube chanel – Youtube.com/aminolabs – or go to this direct link: https://www.youtube.com/watch?v=LEsv0Qvbczs
Necessary safety supplies
Disposable container 500ml-1L
to hold tubes, loops and other contaminated waste (e.g., yogurt container,
plastic cup).| Latex or nitrile gloves
like the ones found at a pharmacy. at least 10 pairs
---|---
Chlorinated bleach spray
1 regular bottle (or you can mix a 10% solution: 1 part bleach to 9 parts
water in a spray bottle)| Bleach ~250 mL
to inactivate all the experiment materials at the end of the experiment.
Timeline
Plate Extract-it Kit
Experiment Protocol
An Experiment Protocol is the scientific way to talk about your instructions
for completing the exercises.
These will not include any theory or background information on the why of each
step. You can find that in the Virtual Bioengineer Simulator and Tutorial
videos.
In the next pages are detailed, step by step instructions to complete the
experiment. Please make sure to read all the steps in the section/sub-sections
prior to starting the hands-on manipulation; some steps will be done in rapid
sequences.
Remember that a series of real-time video tutorials covering our different
kits are available on our youtube channel :
youtube.com/c/AminoLabs
0. Prepare your space
Goal Set yourself up for sucess.
Materials from the kit
(1) Inactivation bag| Materials not in your kit
(1) 1L discard container
Chlorinated bleach spray or wipes| Paper towels
(1/person) Pair of gloves
---|---|---
Make sure you have the necessary materials as explained on page 15, including
gloves, microwave, and cleaner before you start.
0.1 Put on your gloves, and if you have one, your lab coat or apron.
0.2 Set your inactivation bag inside your disposable 1L yogurt-type
container. You will use your inactivation bag to dispose of:
- your tubes of cells if you are not saving them for a future experiment*,
- any used inactivation loops,
- bacteria paint palette once it is used (unless you are saving it to paint your other canvases later)*
- any empty tubes like the agar, buffer and selection tubes,
- any gloves that have touched bacteria.
You can dispose of paper and plastic packaging in the regular garbage can, as
well as gloves if you have not accidentally touched bacteria.
0.3 Wipe down your work surface with the chlorinated bleach spray or
wipes.
0.4 Set down your DNA Playground, BioExplorer, or other personal lab
equipment (it is recommended you use an incubator for this experiment) on or
near your work surface. Make sure it is level and on a stable surface. Refer
to the instruction manual to make sure you know how to use your equipment
safely.
- If you are saving the tubes of cells or your painting palette for a future experiment, place them back in their ziploc bag after use and store them in a refrigerator. We recommend you use a sealed plastic container to store all your experiment materials inside a refrigerator if you also use this to store food or drinks. *
1. Creating selective LB Agar Plates Day 1, 25 minutes
Goal Create selective LB agar plates.
Materials from your kit
(1) 50 mL sterile water
(1) LB agar powder| (1) antibiotic pill
(4) 6 cm petri dishes| Materials not in your kit
(1) Sharpie marker
---|---|---
Prepare
1.1 Label each petri dish with a sharpie-type pen. Make sure to label the
bottom of the petri dishes (the bottom is the part that has the smaller
diameter of the two: the bottom fits inside the lid). Label 4x S. (for
selective) + Add [your initials] if doing this in groups with multiple kits.
Mix the Agar
1.2 Unscrew the lid from the sterile water bottle and keep it loosely on
top of the bottle to prevent any contaminants from entering the water, but
allowing air to escape. This will prevent pressure build-ups.
1.3 Place the bottle in the microwave and heat the water until you see it
boil. You can use 45 seconds as your starting time but you have to see a
rolling boil where many bubbles are rising constantly before you continue to
the next step. Careful, the bottle will be hot! !! If the water does not boil,
the agar powder will not dissolve and your plates will not solidfy !!
1.4 Add the tube of agar powder to the boiling water. Careful, the water
is hot! Some agar powder may “clump” around the lip of the tube due to the
water evaporation. This is okay, we have accounted for this possible loss. 1.5
Microwave the water and agar powder in 4 seconds intervals until you see it
boil again. Instead of a rolling boil, you will see more of a foam forming
above the molten LB agar liquid. Careful, the liquid will boil over if you
microwave in more than 4 sec. increments. After you see the liquid foaming,
swirl to mix for 10 seconds. Try not to shake vigorously as this will create
bubbles in your agar and make the surface of your agar uneven.
Note: If you’ve done the Engineer-it Kit before, note that you will not
be making a non-selective plate. All 4 plates will be selective agar.
Make selective (S.) plates
1.6 Add the antibiotic pill to the bottle of agar and gently swirl for a
few minutes until the contents of the pill have dissolved. Do not introduce
bubbles into the LB agar: don’t swirl too vigorously. The gelatin capsule may
not fully dissolve. The important thing is that the contents of the capsule do
dissolve.
1.7 Once the antibiotic pill is dissolved, pour the molten LB agar into
the bottom half of the 4 petri dishes. Place the lids 3/4 of the way back on
so that the agar can cool and dry (solidify).
Pro-tip: If there are water droplets on the surface of the LB agar, this can
disrupt your art. Bacteria that you will be painting with can enter a droplet
and spread throughout the droplet therefore ‘smudging’ your art. To avoid this
make sure the lid is partially over top to allow for evaporation and a dry LB
agar surface.
1.8 Let the LB agar harden. This can take up to 20 minutes depending on
how warm and humid your environment is. You will use 1 plate in the next step.
You can store the remaining 3 plates in the ziploc bag in the refrigerator for
day 2.
Troubleshooting tip
If your plates do not solidify after 30 minutes it is very likely that the
water was not boiled enough to dissolve the agar powder. As a ‘hack’, you can
pour all of the petri dish content back into the water bottle and microwave
until you see it boil. Swirl to mix and re-pour your plates.
Checkpoint – Agar Plates
Use this guide to check if you are ready to move onto the next step.
|
---|---
A perfect Agar plate is completely clear and solid – if you set it 4” above
some image or text, you should be able to read it / see it clearly.
Move on to the next step!| An agar plate that is cloudy and/or bumpy and/or
soft is not ideal – if you set your plate 4” above some text or image and
cannot see clearly through it, it means you needed more boiling or mixing.
Troubleshooting tip
If your plates do not solidify after 30 minutes it is very likely that the
water was not boiled enough to dissolve the agar powder. As a ‘hack’, you can
pour all of the petri dish content back into the water bottle and microwave
until you see it boil. Swirl to mix and re-pour your plates.
Unfortunately, if the agar does not solidify, this means you need to halt your
experiment and complete the troubleshooting guide and follow the instructions
at www.amino.bio/troubleshoot
2. Amplify (Culture) engineered cells Day 1, 15 minutes + 24 hours wait
time
Goal Create living paintings
Materials from your kit
(2) Selective Agar plates| Your engineered bacteria| (2)Yellow Loops
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Prepare
2.1 If you have an incubator, turn it on to 37°C.
Streak
2.2 Take your engineered cells from your Engineer-it Kit experiment.
Place your petri dish on top of the double zigzag pattern stencil. Take one
yellow loop, pick one or more colonies of engineered cells on your plate. You
pick a colony by touching the end of the loop to it, gently rubbing it.
2.3 With your picked colony(ies) on your loop, trace one of the zig zag
across the fresh selective agar plate.
2.4 Using the same yellow loop, trace the second zigzag, which is at 90°
of the first. This will ensure you will have lots of cells growing across your
plate. Discard the loop.
2.5 Using the same yellow loop, repeat the same exercise on the second
fresh selective agar plate.
Incubate Overnight
2.6 Incubate your streaked plate upside down at ~37°C for up to 24 hours:
Flip your plates upside down so that the agar is up and the lid down. Set your
plate in the incubator or in a plastic bag in a warm location if you do not
have an incubator. Incubate for 16-24 hours (it may take longer if you do not
have an incubator. Bacteria prefer 37°C to grow optimally). If you have Amino
Labs’ minilab, remember to close the incubator door and lock it!
Checkpoint – Did your cultured cells grow?
Use this guide to check if you are ready to move onto the next step.
|
---|---
A perfect cultured plate will have many colonies or a lawn of bacteria on it.
Congratulations!
Move on to the next step.| If you see no growth on your plates, your
amplification of cells may not have worked or you haven’t incubated long
enough. Try to incubate longer.
If you cannot see any growth after 48 hours, repeat step 2 on the 2 unused
plates from step 6. If you still cannot see growth on these after incubating
48 hours, your experiment may have failed. Don’t be discouraged. In science,
failure is a chance to learn more.
Complete the troubleshooting guide at
amino.bio/troubleshoot
3. Harvest & lyse the bacteria Day 2, 15-30 minutes
Goal Suspend the cells in lysis buffer and enzyme in order to break down the cell wall and release the produc
Materials from your kit
(2) Your engineered bacteria plates
(1)Yellow Loop| (1) Lysis Buffer tube
(1) Lysis Accelerator| (1) 1 mL Pipet
---|---|---
Harvest
3.1 Verify if your cells have grown and are fully expressing their
traits. If they have not yet, keep incubating.
If they have, take them out of the incubator.
3.2 Turn on the Cold Station to Ice, and set the tube of Lysis buffer in
the cold station.
3.3 Get your tube of Lysis Accelerator from the freezer and also place it
in the Cold station.
3.4 Take a yellow inoculating loop and gently drag the loop across the
surface of the LB agar to collect the cells from the petri dish. The cells
will collect inside the loop.
3.5 Once the loop is full, dip it into the Lysis buffer tube and twist it
to dislodge and mix in the cells in the buffer.
3.6 Repeat collecting and blending cells in the buffer until you’ve
collected all of the cells on the two petri dishes.
3.7 Using the yellow loop, blend the cells and buffer for a further 60
seconds to make sure they are fully suspended. This will help the surfactant
in the Lysis Buffer begin lysing the cells.
3.8 Open the tube of Lysis Accelerator that you have placed on the Cold
Station. Using one of the plastic pipette included in the your kit, suck up
all of the Lysis Buffer and cells mixture and gently add it into the Lysis
Accelerator tube. You may have to repeat this a few times to get all the
mixture into the Lysis Accelerator tube.
3.9 Leave your empty Lysis Buffer tube in the cold station and place your
pipet inside to hold it until you use it again in the next few steps.
3.10 Once you have moved all the buffer over to the Lysis accelerator
tube, firmly close the lid of the Lysis accelerator tube and vigorously shake
it for 30 seconds to ensure that it is fully mixed.
3.11 Place it on the cold station to cool for 5 minutes.
3.12 After 5 minutes, use your pipette to move all the liquid in the
Lysis Accelerator tube back into the clear Lysis Buffer tube. Be careful as
the liquid will be bubbly. You can wait a few seconds for the bubbles to go
down before putting on the lid.
3.13 Turn off the cold station and let the mixture incubate at room
temperature for 15 minutes, or up to 24 hours.
You can now dispose of the Lysis Accelerator tube and pipette in the
inactivation bag.
4. Collect & Filter-Sterilized bacteria Day 3, 30 minutes + 24 hours wait
time
Goal Passing the extracted pigment through a 0.22 um filter to get rid of
cells and other debris (sterilize the products)
Materials from your kit
Your tube from the previous step
(1) Syringe| (1) Syringe Filter
(1) 1.5 mL tube for final product
---|---
Pellet
4.1 Balance your microcentrifuge according to the manufacturer’s
instruction by adding a similarly weighted tube directly opposite your tube..
The kit provides a balancing tube for your help. This tube includes a volume
of of liquid that should be close to what you have inside your Lysis Buffer
tube (Lysis buffer, cells, Lysis Accelerator). If you have spilled any of your
Lysis buffer and cells, you will need to use the Pipet to remove some liquid.
4.2 Add your tube of Lysis Buffer and cells into the centrifuge. Spin at
maximum speed (13,000 x g to 15,000 xg) for 20 minutes. Refer to the
centrifuge manufacturer’s instruction for additional centrifuging help.
Filter-Sterilize
While your solution is centrifuging, prepare your next step:
4.3 Using your DNA Playground as a tube holder, get the Final Product
Tube, remove the lid and place in on the Hot tube station (in the off
setting).
4.4 Remove the syringe plunger from the syringe and lay it on a clean
surface.
4.5 Open up the syringe filter by taking the paper cover off. DO NOT
fully remove the filter! You want to make sure that you do not contaminate the
other end, the one that will not screw into the syringe but will dispense your
sterilized sample.
4.6 Holding the filter via the plastic container, screw on the syringe to
the filter so it is firmly connected.
4.7 Place the syringe and filter inside the Burst Bag, with the tip of
the filter poking out of the triangle cut at the bottom of the Burst Bag. You
can lay this on the table and be sure not to touch the sterile end of the
filter.
4.8 Once centrifuged, gently pour your centrifuged sample into the open
syringe. Be careful to only pour the liquid. You can also use one of the
plastic pipette to add the liquid to the syringe. If the the pellet of cell
debris falls into the syringe, it will clog the filter! If this happens, and
cell debris gets into the syringe, pour the entire mixture back into your tube
and repeat the centrifugation.
4.9 With the sample in the syringe, hold it so that the sterile end that
will release your filtered solution is pointing into the Final Product Tube.
4.10 Replace the syringe plunger into the syringe and GENTLY press down.
If you have effectively centrifuged your sample, the plunger should slowly
fall until all the solution passes through. Any cell debris or bacteria that
werein the sample will be trapped in the filter, while small molecules such as
your proteins will pass through.
4.11 Close and tighten the lid on your Final Product tube.
Congratulations! The solution in this tube is an extracted, filter-sterilize
product you can now use!
You now have sterilized proteins you microfactured yourself with your genetic
engineering skills! If you extracted color proteins, you can store your final
pigment in the refrigerator, or at room temperature. Many colour pigments will
keep their colour for more than a year if kept out of the sun!
Share your creations with us!
@aminobiolab
Storage, Disposal, Clean Up
After you sees your results, all experiment Petri dishes, tubes of cells
and loops should be in the inactivation bag in your discard container.
Disposing of experiment materials is an integral part of the experiment.
Always wear gloves for cleanup!
A. Preserving Petri dishes: If you want to preserve the living paintings
or experiment results in Petri dishes instead of disposing of them, use one of
our Keep-it kits. This will help you maintain the petri dish by pouring a
special resin on top. If you do not have Keep-it Kits on hand but will be
getting one soon, keep the Petri dishes you want to preserve in a ziploc bag
in a cool area and out of sunlight in the meantime. You can refrigerate it to
keep it “fresh” for up to a month.
B. Reusable materials: If you have DNA in your kit, it can last up to 6
months when stored in a refrigerator. If you wish to keep it, store it in a
ziploc bag inside a sealed plastic container in a refrigerator away from food
items. If you do not wish to keep it, add to an inactivation bag. Make sure
the lids are separate from the tubes so that the inactivating liquid can get
inside. If you see any mold or unknown bacteria growing on any material at any
point, immediately inactivate them by using a solution of bleach water. Follow
the inactivation instructions below. If you are out of inactivation bags, use
a sturdy ziploc type bag or disposable container with a lid. Always wear
gloves when handling experiment materials and cleaners!
C. Unused ingredients: If you did not use all the agar Petri dishes you
poured, store these for later use. Store them in their ziploc bag within a
sealed container in the refrigerator for up to a few months. Keep them away
from food items. If you see any mold or unknown bacteria growing inside, then
you should always immediately inactivate the Petri dishes.
D. Inactivation: Make sure all bacteria, agar, tubes, loops,
paintbrushes, Petri dishes, contaminated gloves, and other non-paper material
you are not keeping are in the inactivation bag. Remember that any paper
packaging like loop wrappers, plastic bags, and gloves that have not touched
bacteria go in the regular garbage or recycling.
Make sure all the tubes have their lids off once in the inactivation bag and
add a solution of 1 part bleach to 4 to 6 parts water to the inactivation bag.
Close the bag and let sit for 24 to 48 hours before discarding the liquid in
the toilet and the solids & bags in the garbage. Step-by-step instructions are
on the inactivation bag and in an Inactivation video on youtube;
youtube.com/c/AminoLabs.
Spray some chlorinated bleach cleaner in the discard container once emptied if
it has become contaminated by experiment materials.
Let it sit for an hour before wiping down. You can wait to wipe it down until
you empty out your inactivation bags the next day.
E. Clean your workspace: Use a chlorinated spray cleaner, wipes, or a
solution of 1 part chlorinated bleach to 9 parts water to wipe down your work
area and equipment. You can wipe down the minilabs with this solution and
follow it with an eyeglass or window cleaner to remove the inevitable
streaking from the bleach cleaner. Never use rubbing alcohol (isopropyl
alcohol) on the DNA Playgrounds.
**Glossary
**
Agar: is a Jello-like substance that serves as a growth media for
bacteria. It is mixed with our bacteria’s favorite food: Lysogeny broth (LB).
LB is made up of yeast, vitamins, and minerals. LB can also be found liquid-
form.
Antibiotics: When you transform bacteria, they will become resistant to a
type of antibiotics no longer used in hospitals. This antibiotic will be mixed
in with the agar and LB so that, as you incubate your culture, only
transformed bacteria will grow. This is called a “selection marker”.
Autoclave: An autoclave is a machine used to carry out industrial and
scientific processes requiring elevated temperature and pressure in relation
to ambient pressure/temperature. In life science, autoclaves are used to
sterilize equipment and supplies by subjecting them to pressurized saturated
steam at high temperatures (around 250 °F) for several minutes, up to an hour.
Autoclaves are similar to some baby bottle sterilizers which you might be
familiar with.
Buffers: Buffers are saline solutions that help, in this case, open up
the cell membranes so that they may take up new DNA.
Cells: Cells are tiny, living units that function like mini-factories.
Bacteria are single-celled organisms (unicellular) microorganisms. They are
different from plant and animal cells because they don’t have a distinct,
membrane-enclosed nucleus containing genetic material. Instead, their DNA
floats in a tan- gle inside the cell. Individual bacteria can only be seen
with a microscope, but they reproduce so rap- idly that they often form
colonies that we can see. Bacteria reproduce when one cell splits into two
cells through a process called binary fission. Fission occurs rapidly, in as
little as 20 minutes.
Competent Cells: Since DNA is a very hydrophilic molecule, it won’t
normally pass through a bacterial cell’s membrane. In order to make bacteria
take in the DNA plasmid, the cells must first be made “competent” to take up
DNA. This is done by creating small holes in the bacterial cells by suspending
them in a solution with a high concentration of calcium (the transformation
buffer). DNA can then be forced into the cells by incubating the cells and the
DNA together on ice, placing them briefly at 42°C (heat shock), and then
putting them back on ice. This causes the bacteria to take in the DNA and is
called “Transformation”.
DNA: The DNA is the set of instructions that tell the cell how to
function like a computer program tells your computer what to do. DNA stands
for Deoxyribonucleic acid.
DNA plasmid: A plasmid is a small circular piece of DNA (about 2,000 to
10,000 base pairs) that contains essential genetic information for the growth
of bacteria. Bacteria share vital information by passing it among themselves
in the form of genes in plasmids. By inserting a new plasmid in our bacteria,
we can get them to produce things for us,can get them to produce things for
us, ike mini-factories. In this case, we have a plasmid that encodes for the
creation of colorful pigments.
Genome: a genome is all genetic material of an organism. It consists of
DNA. Learn more about genomes in the What is DNA? simulator on amino.bio
Heatshock: is when the cells are moved from icecold to warm temperature,
typically 42°C, to take in DNA plasmids more efficiently.
Inoculation: is when you introduce bacteria into a medium suitable for
its growth.
Inoculating Loops: are used to transfer liquids, cells, and DNA from one
vial to the next instead of traditional lab pipettes, making your job easier,
and less costly. They come in different pre-calibrated sizes, so you do not
need to worry about minuscule liquid volumes. They are also used to spread
bacteria on an agar surface without puncturing the soft agar.
Non-Selective: A non-selective plate means that any cells/bacteria put on
this agar will grow as long as they are oxygen-loving organisms (called
aerobic bacteria).
Plates (or Petri dish): A petri dish is a small plastic container used to
culture (grow) bacteria in a controlled environment.
Recovery period: is the period after the heat shock in which the cells
develop their antibiotics resistance and start dividing.
Selective: A selective plate means it contains antibiotics. When you
insert a new DNA program into cells to make them create pigments, or anything
else, you also put a “selective marker” (antibiotics resistance) inside the
code. This means that only the cells that have taken up the new program will
be able to grow on a plate that has the antibiotics mixed in. You only get the
cells you transformed!
Transformation: See competent cells.
Troubleshooting
Here are some possible common issues:
Your agar is too wet/ doesn’t solidify:
When done correctly, the agar will be the consistency of Jell-O.
If it is not:
- You likely did not heat (boil) the water before, or after adding the LB agar powder
- You might not have added all the powder from the tube, resulting in too much water vs. LB agar powder.
- You may not have fully dissolved the powder, meaning it cannot turn into a gel and will look cloudy. You can practice by making Jell-O! Next time heat and swirl longer to ensure the powder is fully dissolved.
You don’t have any colonies and its been 24+ hours:
Don’t worry, every scientist has experienced this, and it can take some
practice before success.
-
Double check that your incubator is on at 37°C. If it is not, or if you are growing at room temperature, then it can take much longer to see the bacteria colonies. Keep waiting!
If you kept the second half of your recovered cells, you can pour them on your plate after 48 hours of seeing no engineered colonies grow and keep incubating. -
You may need to try again to hone your skills. See our Youtube videos for tips and tricks on how to improve your chances of success.
Your colonies of bacteria grew, but they are the wrong color or there is
mold on your petri dish:
Danger! If at the end of, or during, the incubation period your resulting
bacteria/plate is: a)not the right color; b)is black when it shouldn’t be,
this is a sign that your culture is NOT YOUR ENGINEERED BACTERIA. You should
immediately inactivate it and clean your space and unit.
To inactivate it, either add it to the inactivation bag or pour 100%
chlorinated bleach into the dish, put the lid on and let it sit for 24 hours
before throwing it out: The strong oxidizing environment degrades any living
organisms. After 24 hours, if there are still organisms present add more
concentrated bleach until it is almost full, and let stand for a further 24
hours.
There may be mold in your environment. We recommend, getting a small air
purifier with a HEPA filter for the room.
Always be aware that concentrated bleach is a strong oxidizing agent and if poured on the skin can cause irritation, and on clothes remove color. Follow the safety and handling protocol on the manufacturer’s label.
Find an interactive troubleshooter online at
amino.bio/troubleshoot. We recommend
using it for tips, tricks and to claim your Success Guarantee Kit if you need
of one.
If anything else causes you issues, please contact us :
help@amino.bio
More Information
| All Amino Labs products, from the hardware to the DNA, are invented,
designed, manufactured and shipped by us, in our laboratory- workshop in
Canada and we’d love to hear your feedback and suggestions to continue to make
our products better and fitting to your needs. Answers to your questions and
help are also just an email away.
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| Help and General inquiries: help@amino.bio
Feedback, Suggestions, Comments: info@amino.bio
Documents / Resources
|
amino labs Extract it Kit Extract Biopigments from
Bacteria
[pdf] User Manual
Extract it Kit Extract Biopigments from Bacteria, Extract, it Kit Extract
Biopigments from Bacteria, Extract Biopigments from Bacteria, Biopigments from
Bacteria, Bacteria
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Read User Manual Online (PDF format)
Read User Manual Online (PDF format) >>