miniPCR bioTM Learning Lab BioBits®: Central Dogma

miniPCR bio

Learning Lab

BioBits®:

Central

Dogma

miniPCR bio

Learning Lab

BioBits

: Central Dogma

Instructor’s and Student’s Guide

Version: 1.2

Release: July 2020

miniPCR bio

Learning Lab. BioBits

: Central Dogma

P./2

Instructor’s Guide

Overview P.03

Materials needed P.04

Preparatory activities P.05

Student’s Guide P.06

 Backgroundandsignicance     P.07

Today’s lab P.11

Laboratory guide P.13

Study questions P.20

Expected results P.25

Additional supports and extension activities P.30

Teacher’s notes and placement in unit P.38

Learning goals and skills developed P.39

Standards alignment P.40

Ordering information P.41

About miniPCR bio

Learning Labs and the BioBits

system P.42

Instructor’s

Guide Contents

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Instructor’s Guide

Preparatory activities

(Instructor)

Class-time activities

(Students)

Portion DNA, water,

kanamycin and BioBits

pellets for each group

10 min.

A. Setup of BioBits® reactions

10 min.

B. Incubation and initial observations

20 min.

C. Final observation

10 min.

Overview

At a glance

With minimal equipment requirements and a quick and straightforward protocol, students will use

BioBits

reactions to visualize the flow of genetic information and monitor transcription and translation

in real-time through fluorescence. This activity serves as an excellent interactive tool for learning the

central dogma of molecular biology and exposes students to cutting-edge synthetic biology.

BioBits

cell-free reactions are tiny molecular factories that can create a variety of proteins, from brightly

colored fluorescent proteins to functional enzymes, without the need for cell culture. When dry, BioBits

pellets are dormant, but they can be activated by simply adding water. Researchers have been using cell-

free reactions in their laboratories for years, with applications ranging from novel therapeutic discovery

to field diagnostics. Now the BioBits

cell-free system makes this cutting-edge technology accessible

anywhere to anyone interested in learning molecular biology and is an excellent teaching tool to enhance

biology education both within and beyond the classroom.

TIME TECHNIQUES TOPICS LEVEL

MIN.

Micropipetting

Cell-free protein

synthesis

Two class periods:

A. Initial set up and observations:

30 minutes.

B. Final observations: 10 minutes.

Period B should be conducted

between8and72hoursafterA.

Central dogma of

molecular biology

Protein synthesis

Transcription

and translation

Gene expression

Fluorescence.

Introductory

Advanced

Intermediate

MIN.*

MIN.

*Finalobservationcanbedoneanytimebetween8to72hoursafterthefirstclassperiod(typically24hours).

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Instructor’s Guide

Reagents

and supplies

Amount

provided

in kit

Amount

needed

per lab

group

Storage

Teacher’s

checklist

BioBits

pellets in PCR strip

tubes

• Keep BioBits

in the

vacuum-sealedpouchat-20˚C

as long as possible

Four 8-tube strips

to be broken into

strips of 4 tubes

1 strip of

four tubes

-20˚C

Nuclease-Free Water 200 µl 20 µl

-20˚C

DNA A 150 µl 15 µl

-20˚C

DNA B 100 µl 10 µl

-20˚C

Kanamycin 100 µl 10 µl

-20˚C

Plastic tubes: to aliquot reagents

•Anysize0.2–1.7mL

37°C heat source

• e.g., miniPCR

machine,

incubator, water bath

• Body heat works as well

(equipment

can also

be shared

between

groups)

P51™ molecular uorescence

viewer or other blue light

Illuminator:

e.g., blueGel™ or blueBox™.

The illuminator needs to have an

orange(notyellow)lter.

(equipment

can also

be shared

between

groups)

Micropipettes: 2-20μlare

recommended

Disposable micropipette tips

10+

Other supplies:

• Disposable laboratory gloves

• Protective eyewear

• Permanent marker

• Optional: tube rack

Materials needed

Supplied in kitSupplied by teacher

Storage notes: Pellets can be stored for up to 6 months and DNA and kanamycin for 12 months from date of

receipt when stored at -20 °C. Store any unused pellets in an airtight bag with the supplied orange desiccant card.

Iffreezerstorageisnotavailable,thereagentsmaybestoredinthefridge(approx.4˚C).Pelletsareviableforup

to three months from the date of receipt when stored in the fridge. DNA and kanamycin can be stored for up to

six months when kept in the fridge.

Thereagentssuppliedinthekitaresufficientfor8labgroups(recommendedlabgroupsize:2-4).

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Instructor’s Guide

Preparatory activities

Distributing reagents and supplies

• For each lab group, label and dispense in separate microtubes:

- DNA A Label as “A” 15 μL

- DNA B Label as “B” 10 μL

-Water(Nuclease-Free)  Labelas“W”  20μL

- Kanamycin Label as “K” 10 μL

• Each lab group will additionally need the following supplies:

- 1 strip of 4 tubes of BioBits

pellets. Separate each strip of 8-tubes in half to

create 4-tube strips.

• Recommended method is by razor blade to cleanly cut the strip in half.

Scissors will also work.

-Micropipettes,onepergroup(2-20μLrangeisrecommended).

-Disposablemicropipettetips(atleast10pergroup)andasmallbeaker/cupto

dispose of them.

-Permanentmarker(ideallyfine-tipped).

- Access to a P51

molecular fluorescence viewer or other blue light illuminator

(withanorange—not yellow—filter).

-Accesstoa37°Cheatsource(bodyheatworkswellifotherheatsourcenot

available).

MIN.

The following activities should be carried out by the instructor ahead of class.

P./6

Student’s Guide

Backgroundandsignicance      P.07

Today’s lab P.11

Laboratory guide P.13

Study questions P.20

Student’s

Guide Contents

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: Central Dogma

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Background and significance

Overview

Today, you will be using cutting-edge BioBits

cell-free technology to explore the world of synthetic

biology. Each BioBits

pellet contains all the necessary reagents and cellular components required

to perform transcription and translation without cells —all you need to do is simply add water and

your DNA of interest. Researchers use these kinds of cell-free reactions to develop new therapeutics,

medical diagnostics, and more. The BioBits

cell-system allows anyone interested in tinkering with

biology to make proteins anywhere.

In today’s activity you will use the BioBits

cell-free system to visualize the flow of genetic

information and monitor the processes of transcription and translation in real time.

Protein synthesis is usually carried out inside living cells, but BioBits

pellets allow this process to be

carried out without cells. Using DNA that encodes for green and red fluorescent markers, you will be

able to observe both the production of RNA and of protein as they occur in real time. You will also

explore ways to interrupt specific steps in the molecular flow of information from DNA

to protein.

The central dogma of molecular biology

In1957,FrancisCrick,oneofthediscoverersofthestructureofDNA,gavealecturethatprofoundly

influenced how biologists think about genetic information and molecular biology in general. It was

only four years after the discovery of DNA structure, but it was already well accepted that DNA was

the molecule of heredity. Yet the details of how DNA actually encoded genetic information and what

thatinformationencodedforwerestilllargelyuncertain.This1957lectureproposedaconceptual

framework for how the system most likely functions based on the little data that was available then.

Crick called this framework the central dogma of molecular biology.

Dogma is a term typically used to convey an idea that is so fundamental to a field that its truth is

undebatable. Crick chose this word because he felt so strongly that his central idea must be correct,

even though the evidence available at the time was scant. When we use the term central dogma

today, we do not mean to imply that this framework must be accepted unquestioningly. Instead,

dogma conveys that this idea is so fundamental to understanding life and heredity that, in order to

understand molecular biology, one must first comprehend this central principle.

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What was this realization that is now so central to

the understanding of all molecular biology? Crick

outlined the process by which the instructions

contained in DNA are transformed into cellular

function. Simply stated, the central dogma of

molecular biology explains the flow of genetic

information whereby DNA is able to code for

RNA and RNA is able to code for protein. More

specifically, it says that DNA and RNA can both

store and transfer genetic information needed to

make proteins, whereas protein cannot store the

informationneededtomakeDNAorRNA(Figure1).

In Crick’s words, “the main function of the genetic

Figure 1: One schematic representation of the central

dogma of molecular biology. DNA information can be

transferred to RNA and used to make proteins, but

proteins cannot store the information needed to make

DNA, RNA or protein.

material is to control … the synthesis of proteins”. And “once information has got into a protein,

it can’t get out again.” These predictions were strikingly accurate —to the point that his lecture

even accurately predicted the existence of yet undiscovered molecules necessary to turn genetic

information into functional products.

Scientists have since discovered the molecules involved in the flow of genetic information, and have

worked out in great detail how the information in DNA is processed by the cell. But still, more than

sixty years later, as modern research continues to shed new light on the flow of genetic information

within a cell, the central dogma remains the foundation for understanding the relationships between

DNA, RNA and protein.

Proteins: the tools of life

A large number of the molecules carrying out most of life’s essential functions are proteins. As Crick

argued, the major role of DNA is to provide the instructions on how to produce these proteins.

Proteins are made by linking smaller building blocks called amino acids together in a long chain. That

chain then folds into a unique three-dimensional structure. What makes one protein different from

another —whether it breaks down the starches that you eat like amylase does, provides structure to

your cells like tubulin, or moves your muscles like actin and myosin —is the order of the amino acids

in that chain. The main information stored by DNA is simply the order of those amino acids for each

unique protein. The cellular machinery has the ability to read DNA, transfer DNA information into

RNA, and to build the correct amino acid sequences, but cannot reverse the process to read protein

to produce a DNA or RNA sequence.

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How to make a protein

We now think of the flow of information from DNA to protein as a two-step process: transcription

—the production of RNA from a DNA sequence, and translation — the production of protein from an

RNA sequence.

Like protein, DNA consists of building blocks arranged in a long chain. The four building blocks

ofDNAarecallednucleotidesandnamedadenine(A),thymine(T),guanine(G),andcytosine(C).

These nucleotides are arranged in a specific order and connected into a long double helix. Within

this double helix are sequences of nucleotides that contain the information to make different

proteins. The exact structure of this information can vary across classes of organisms, but there

are some commonalities throughout: a promoter sequence that tells the cellular machinery where

the information begins, a protein coding sequence that contains the information that determines

the order of the amino acids, and finally a signal that marks where the transcribed information

ends. In prokaryotic systems, like the one you will be working with in this lab, we call this signal the

terminator sequence.

Figure 2: Transcription —the process of copying information from DNA to RNA.

RNA polymerase moves down the DNA sequence and joins ribonucleotides

together into mRNA.

To start the process, the genetic

information stored in DNA is

first transferred into a temporary

copy called messenger RNA

or mRNA. We call the process

of copying DNA to RNA

transcription. Transcription

starts when the RNA polymerase

recognizes a promoter. The

RNA polymerase binds to

the promoter sequence and

begins moving down the DNA,

unwinding the double helix as

it goes. As the RNA polymerase

travels, it joins the building

blocks of RNA into a long strand

(Figure2).ThefourbuildingblocksofRNAarecalledribonucleotidesandnamedadenine(A),uracil

(U),guanine(G)andcytosine(C).Theribonucleotides are structurally similar to DNA nucleotides

and their correct order is determined by pairing the ribonucleotides to their complementary DNA

sequence. RNA polymerase links together the unbound ribonucleotides to the growing single strand

of RNA with energy from ATP driving the reaction.

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The information now stored in the mRNA will

next be read to make a protein in a process

called translation. Reading the information

encoded in the mRNA takes place at the

ribosome. The mRNA binds to and starts

being fed through the ribosome. As the mRNA

moves through the ribosome, the order of

nucleotides is read in groups of three known as

codons —you can think of the nucleotides as

letters, and the codons as words made up of

three nucleotide letters. The start of the protein

coding sequence is marked by a special order

of three nucleotides known as the start codon.

To read the coding sequence in the mRNA,

a different kind of RNA called transfer RNA

or tRNA, binds to the mRNA. On one end of

the tRNA are three nucleotides that pair with

the codon on the mRNA by the rules of base

pairing. On the other end of the tRNA is the

amino acid that is specific to the codon on

that tRNA. As the tRNAs bind to and translate

the information in the mRNA, the ribosome

links the corresponding amino acid to the one

before it, creating the chain that will become

the protein. Just like in transcription, translation

isfueledbyATPasanenergysource(Figure3).

Figure 3: Translation —the process of making proteins from RNA

information. At the ribosome, the mRNA is read by complementary

tRNAs and each tRNA’s corresponding amino acid is linked into the

growing protein chain.

The end of the protein coding sequence is

marked by a stop codon; upon reaching this

codon, the ribosome will release the newly

formed amino acid chain, which will continue

to fold into its final three-dimensional structure.

This new protein may do any of countless

functions depending on its sequence of

amino acids. But regardless of what protein is

Transcription and translation analogy

• When you are copying something word for word,

like writing down an exact quote from an interview,

you are transcribing that information. The process

of transcribing is known as transcription. When RNA

is produced, the order of the bases in the RNA will

resemble an exact copy of one of the DNA strands the

RNA was copied from. For this reason, scientists call this

process transcription. The main difference between the

RNA transcript and the original DNA is that RNA uses

ribonucleotides, where the base uracil is substituted for

the structurally similar thymine.

• When you are copying something in one language into

another, say from English to Spanish, you say you are

translating that information. The process of translating

is known as translation. At the ribosome, mRNA

nucleotides are read to make a sequence of amino

acids, the building blocks of protein. You can think of it

as the language of nucleotides being translated into the

language of amino acids. For this reason, scientists call

this process translation.

made or what organism the process occurs in, whether it be a bacterium or a whale, an amoeba or an oak tree,

the same basic process is followed. DNA is transcribed into RNA by RNA polymerase using ribonucleotides as

building blocks and ATP as an energy source. RNA is then translated at the ribosome, with tRNAs deciphering

the genetic code, bringing amino acids that are joined together into a chain, again with ATP fueling the reaction.

Despite all the complexities that have been discovered in modern molecular biology there is no evidence that the

process can start with protein and go the other way, just as Crick predicted.

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Today’s lab

Transcription and translation without cells

Lab setup

In this lab, you will use DNA that encodes the information for making a fluorescent protein.

Fluorescentproteinscanbefoundinorganismssuchasjellyfishorcoralandwilllightup(fluoresce)

when exposed to a specific wavelength of light, usually blue or UV light. In nature, it is hypothesized

that organisms use fluorescent proteins to ward off predators or attract prey. In research, scientists

use these same fluorescent proteins as visual markers or signals that illuminate what is happening

in their experiments. Similarly, today you will be using fluorescence to track the flow of genetic

information as the DNA is transcribed to RNA, and the RNA is translated to protein.

You will be given a sample of DNA containing a gene with the information to make a red fluorescent

protein. When the gene is transcribed and then translated, you will be able to visually confirm the

presence of the protein by observing red fluorescence. However, observing the presence of mRNA

after transcription is usually more difficult because mRNA is not typically visible to the naked eye.

For this lab, we will be able to visualize mRNA with a unique genetic feature built into this gene.

Just upstream of the coding sequence the gene encodes an aptamer, a specially designed sequence

of nucleotides that has the ability to selectively bind to other molecules when transcribed. BioBits

pellets contain a specific chemical that this aptamer will bind to, and when it does, it will emit

Figure 4: Essential cellular machinery can be

extracted from cells and supplemented with

molecular building blocks and energy to create

a cell-free system that is still capable of carrying

out transcription and translation.

Transcription and translation typically happen

inside the cells of living organisms. But it is possible

to perform these processes in a synthetic system

without cells. The BioBits

system you will be using

today is an example of such a system. BioBits

pellets

contain all of the necessary cellular components, such

as RNA polymerases for transcription and ribosomes

for translation. They also contain the required

building blocks —the nucleotides to build mRNA

and the amino acids to build proteins. Furthermore,

they contain ATP, the energy source that powers the

reactions(Figure4).AnyDNAcarryingaproperly

structured protein-coding gene that is added to

the system will result in the synthesis of the protein

encoded by the DNA. In this way,

we can make proteins quickly and easily without any

of the difficulties of culturing live organisms.

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green fluorescence. Hence, green fluorescence will signal transcription, the synthesis of mRNA

from DNA. In this way, using both the green RNA aptamer and the red fluorescent protein as visual

readouts, you will know that you have successfully transcribed DNA to RNA when you see the

green fluorescence and that you have successfully translated RNA to protein when you see the red

fluorescence(Figure5).

Figure 5: The structure of the DNA sequence you will be using in this lab, including the aptamer sequence for the green

RNA signal and the protein coding sequence for the red fluorescent protein.

You will perform four reactions that allow you to investigate the central dogma. The first reaction

will serve as a negative control, where you will add water instead of DNA. To your second reaction,

you will add the DNA sample described and shown above. You will add this same DNA to your third

reaction, but you will also add kanamycin, an antibiotic drug that interferes with ribosome function.

In your fourth reaction, you will add a different DNA sample. Your job, based on your knowledge of

the central dogma, is to predict what you will observe in the first three reactions and then deduce

what occurred in the fourth reaction based on your observations.

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Tube 1 Tube 2 Tube 3 Tube 4

DNA

none 5μlDNAA(A) 5μlDNAA(A) 5μlDNAB(B)

Laboratory guide

A. Setup of BioBits® reactions

You will investigate three samples and one negative control using the BioBits

cell-free system.

1. Label each tube in your strip of four BioBits® pellets on the side, not cap, of the tube

• Label the tubes 1 through 4

•Labelagroupname/symbolsomewhereonthetubes

• Tube 1 will be for your negative control

• Tube 2 will be for your reference reaction

• Tube 3 and 4 will be for your experimental reactions

- You will know what you added to tube 3 and will have to predict the result.

- You will not know what you added to tube 4 and will have to deduce what

you added based on the result.

2. Uncapping BioBits® strip tubes

• Gently tap tubes on the table to collect pellets at the bottom.

• To open tubes, CAREFULLY remove each cap in the strip one at a time,

taking care not to dislodge BioBits

pellets.

3. Add DNA to each BioBits® pellet in the strip. Use a new tip for each sample.

• Use a micropipette to add the DNA solution to dissolve the pellet.

- Do not use the second stop on the pipette.

- Do not yet add liquid to tube 1.

- Add 5 μlofDNAA(A)totubes2and3.

- Add 5 μlofDNAB(B)totube4.

Do not touch your pipette tip to the pellet or the pellet may get stuck inside

the tip. Instead, it may help to touch the pipette tip to the side of the tube so

the DNA is added down the side of the tube, and then to tap the tube so the liquid collects

at the bottom of the tube and dissolves the pellet.

Because the reaction volumes are so small, you want to avoid bubble formation.

We advise against using the second stop on your micropipette, and also against pipetting

up and down to mix.

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4. Pipette the additional reagents to each tube. Use a new tip for each sample.

•Add7μLofwater(W)totube1.

• Add 2 μLofkanamycin(K)totube3.

• Add 2 μLofwater(W)totubes2and4.

• Use a micropipette to add the reagents. Do not use the second stop on the pipette.

Tube 1 Tube 2 Tube 3 Tube 4

Reagent

7μlwater

(W) 2μlwater(W)

2μlkan(K)

2μlwater

(W)

5. Close the caps on the tubes.

• You should feel the caps “click” into place if they are closed correctly.

• Make sure all the liquid volume has dissolved the pellet and collects at the bottom of the

tube.

• If necessary, shake down with a flick of the wrist or spin briefly in a microcentrifuge.

6. Immediately observe your tubes in the P51™ viewer or other blue light illuminator.

• Make sure the blue light is on and that an orange filter is in place.

• Dim ambient lights as needed for proper observation.

• Record your observations in Table 2(page18)inthe“Time0“row.

P. 10

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B. Incubation and initial observations

1. Place the tubes at 37°C.

•UseaminiPCR™machinesettoheatblockmodeora37°Cincubator.

•Ifyoudon’thaveaminiPCR™machineorotherheatsource,youcanusebodyheat(i.e.,your

hands,underthearm,inyourpocket)towarmthetubes.

• If you have not yet done so, predict what you will see in Table 1(page17)belowduringthe15

minutes of incubation. You can also complete the pre-lab activity and questions in the Study

questionssectionbelow(page20).

2. After 15 minutes, observe your tubes in the P51™ viewer or other blue light illuminator.

• Make sure an orange filter is in place.

• Dim ambient lights as needed for proper observation.

• Recording your observations in Table 2 (page18)inthe“15minutes”row.

3. Store tubes at room temperature.

• The rest of the reaction will occur overnight at room temperature.

• You can leave the tubes in a tube rack or laying flat on the lab bench or table.

• If you have a longer class period, you can continue observing your tubes at additional

time points and record your observations.

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C. Final observation

1. The next class time, observe your tubes in the P51™ viewer or other blue light illuminator.

• Day 2 observation is usually done at approximately 24 hours after Day 1, but can be

doneanytimebetween8hoursto72hoursafterDay1.

• Make sure the blue light is filtered out with an orange filter.

• Dim ambient lights as needed for proper observation.

• Record your observations in Table 2(page18)inthe“Day2”row.

• In Table 3(page18),compareyourTable1predictionsandTable2observations.

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Observation tables and questions

While you are waiting for your tubes to incubate, predict the colors of the reaction in tubes 1 through

3 and explain your thinking in one or two sentences. Tube 4 will be analyzed separately after you

make your observations. Use the Background section of this lab and your concept map forhelp(page20).

Table 1: Predictions

Time 0

Prediction:

Justication:

Prediction:

Justication:

Prediction:

Justication:

Prediction:

Justication:

Prediction:

Justication:

Prediction:

Justication:

Prediction:

Justication:

Prediction:

Justication:

Prediction:

Justication:

15 minutes

Day 2

Tube 1 Tube 2 Tube 3

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Student’s Guide

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Basedonthecolorsyouobserved,statewhetherornotyouthinktranscriptionand/ortranslation

happened in each of the tubes 1-4. Then explain whether this conclusion agrees with your initial

predictions. If it does not, can you think of a reason that it may not?

Transcription? Translation? Do your conclusions match your

predictions from Table 1?

Table 3: What processes occured?

Time 0

15 minutes

Day 2

Note the color of the reaction in each tube at each observation time point.

Tube 1 Tube 2 Tube 3 Tube 4

Table 2: Observations

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Student’s Guide

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CER Table

Fill in the table based on your results from the lab.

What do you think is the most likely explanation for your observations in tube 4?

Claim

Make a clear statement

that answers the above question

(Hint:Focusonwhatyoudidto

make this tube different from the

othertubes.)

Evidence

Provide data from the lab

that supports your claim

Reasoning

Explain clealry why the data you

presented supports your claim.

Includetheunderlyingscientic

principles that link your evidence

to your claim

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: Central Dogma

Student’s Guide

P./20

Study questions

Questions before experiment

Concept map: Processes and components involved in the central dogma

Using the provided word bank, fill in the blanks on this concept map to show the correct flow of

genetic information and the processes involved. Then, use the table below to list the molecular

componentsinvolvedineachprocessanddescribetheirrole/function.Youdonotneedtofillin

every row of each table. Some words may be used more than once.

Component ComponentRole Role

Word bank:

DNA Transcription Translation RNA Polymerase Ribonucleotides mRNA

Ribosome Amino acids Protein ATP tRNAs

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Student’s Guide

P./21

Structure of the gene used in today’s lab

Think about the processes of transcription and translation and how they relate to the gene structure

shown below.

1. Look at the diagram above. If your only goal were to design a sequence that could be transcribed

(regardlessofwhetheritcouldbetranslated),whichpartsofthisdiagramwouldbemostimportant?

Which parts could you get rid of and still have transcription occur? Justify your answer.

2. Refer to the diagram again. Draw a similar diagram here showing what the mRNA would look like.

Only include those features that you think would be transcribed to the mRNA.

Most important

Which features of the original diagram did you not include in your diagram of mRNA?

Explain why you did not include them.

Justification

Could get rid of

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Student’s Guide

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Questions for after the experiment

1. In this experiment, tube 1 was a negative control. Why is having a negative control important for

your experiment?

2. If tube 1 was not included in this lab, what incorrect conclusions could a person make?

3. The central dogma states that genetic information can be passed from DNA to protein, but not

intheotherdirection.Whichreaction(tubenumber1,2,3,or4)mostclearlydemonstratesthat

information was passed from DNA to protein? Explain your answer using evidence from the lab.

4. You added kanamycin, an antibiotic, to tube 3. In bacteria, kanamycin interferes with the ribosomes

and causes mistranslation of proteins. Do your results support this? Explain why or why not.

3. In today’s lab, we have visual markers to know if transcription and translation have occurred.

a. mRNA is not usually fluorescent. Explain how in this lab green fluorescence indicates

transcription has occurred. Include a small drawing or diagram in your answer.

b. Explain how red fluorescence indicates that translation has occurred. Include a small

drawing or diagram in your answer.

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Student’s Guide

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b)Thecodingsequencedoesnothavepromoterbeforeit.

c)Thecodingsequencedoesnothaveastopcodon.

d)Thereisapromoterandacodingsequencebutnoterminatorsequence.

e)Thereisapromoterandaterminatorsequence,butnocodingsequence.

f)NoATPisaddedtotheBioBits

pellet.

g)Thereisnoaptamersequence,butnothingelseischanged.

a)Thecodingsequencedoesnothaveastartcodon.

Example:

a. The presence of a promoter and terminator sequea. The presence of a promoter and terminator sequence means nce means

the mRNA would be produced correctly, but without the mRNA would be produced correctly, but without a start codon a start codon

the ribosome would never start translation properlthe ribosome would never start translation properly.y.

5. For each of the following statements, predict what you think might happen. For each statement

circlethetubeifyouthinkitwouldproduceRNA(andfluorescegreen)and/orprotein(and

fluorescered).IfyoudonotthinkitwillproduceRNAand/orproteincrossthetubeoutandthen

justify your answer in 1-2 sentences. Refer back to the gene structure below to help guide your

answers. You may not know for sure what will happen in each situation, but give the answer you

think is most likely. The first one has been done for you.

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Advanced questions

1. This lab was performed in a cell-free BioBits

reaction. Both aptamers and fluorescent proteins can

be used in living cells as well. What parts of this activity do you think would be much more difficult

in a live cell system?

2. Can you think of anything that could be easier in a live cell system that may be more difficult or is

not possible in this cell-free system?

3. mRNA as an intermediary between DNA and protein.

a. What might be the advantages of using mRNA as an intermediary between DNA and

protein?

b. Imagine a system in which DNA could be translated directly into protein. Can you think of

any advantages of a system that did not use an intermediate molecule? If so, what might they be?

4. Scientists believe the earliest forms of life did not have both DNA and RNA, but they were able

to make proteins. Based on the processes described here, which do you think evolved first, DNA or

RNA? In other words, which molecule could you more easily imagine a cell living without and still

being able to make protein?

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Additional

Supports

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Student’s Guide

Expected

Results

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Expected Results

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Expected results

Listed below are expected results for each reaction accompanied by a brief explanation. Note that

exact timing of results may differ based on incubation temperature or other factors, but the overall

trends should not change. You may also observe small differences in brightness across different

groups which can arise from variability in micropipetting and sample handling.

Tube 1 (Negative control):

Students added only water with no DNA to tube 1, so no green or red fluorescence is expected

throughout the course of the experiment. Students may detect low levels of autofluorescence, as

the components in BioBits

pellets will show low levels of fluorescence on their own. This low level

of autofluorescence can be used to demonstrate the importance of including negative controls in

experiments. Absent a negative control, baseline levels of autofluorescence may be mistaken for a

true reaction product.

Tube 2 (Reference reaction):

Students added DNA A, encoding both the RNA aptamer sequence and the coding sequence for a

red fluorescent protein. Students can expect observable green fluorescence after ~15-30 minutes,

arising from the RNA aptamer and indicating the presence of messenger RNA. Red fluorescence,

indicating the presence of red fluorescent protein, should become visible after three to four hours

and reach full fluorescence after approximately eight hours. The red fluorescent protein will remain

visible for at least 1-2 weeks.

Tube 3 (Experimental reaction - students predict the result):

Students added DNA A, encoding both the RNA aptamer sequence and the coding sequence for a

red fluorescent protein. They then added the translational inhibitor kanamycin. Students can expect

toobservegreenfluorescenceafter~15-30minutes,indicatingthepresenceofmessengerRNA(as

intube2).ThisgreenfluorescencearisingfromtheRNAaptamerwillfadeafterthreetofourhours

and will not be visible the following day. This is because RNA is typically transient and degrades

overtime(foramoredetailedexplanation,see“Additionalobservationsandexplanations”below).

Students are not expected to observe any red fluorescence. This is because kanamycin will interfere

with translation by binding to the ribosome, causing misalignment of the mRNA during translation.

This will result in non-functional mistranslated protein, or no protein at all.

Tube 4 (Experimental reaction)

Students added DNA B to tube 4. DNA B contains a promoter sequence, the RNA aptamer

sequence, and a transcription termination sequence. DNA B does not contain a protein coding

sequence. Students should observe green fluorescence after ~15-30 minutes, indicating the presence

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ofRNA(asintube2and3).Thisgreenfluorescencewillfadeafterfivetosixhoursandwillnotbe

visible when viewed the following day. This is because RNA is typically not stable in solution and is

expectedtodegradeovertime(foramoredetailedexplanation,see“Additionalobservationsand

explanations”below).SinceDNABismissingaproteincodingsequence,studentswillnotobserve

any red fluorescence as no protein is being translated.

Students should be able to reach the conclusion that there has been some change to the coding

sequence of the DNA, but they will not have enough information to definitively conclude that the

coding sequence is missing entirely. Other reasonable explanations that students may suggest for

what occurred in tube 4 include but are not limited to:

• DNA B has a mutation in the protein coding sequence that makes the protein non-functional.

• DNA B is missing either start codon or a stop codon.

• DNA B has a mutation that introduces a premature stop codon.

Additional observations and explanations

Observations concerning green fluorescence:

Intubes2,3,and4,thegreenfluorescence(indicatingmessengerRNA)willbevisibleafter~15-30

minutes, but will begin to fade after a few hours and will no longer be visible the next day. This is

because messenger RNA is not stable and will begin to break down in solution over time, likely due

to the action of RNases present in the BioBits

pellet. Furthermore, the fluorescent RNA aptamer is

selectedforoptimalfunctionat37°C.ThisiswhytheBioBits

reactionsmustbeincubatedat37°C

before initially viewing the green fluorescence. Changing the temperature of the reaction may affect

the function of the RNA aptamer, thus reducing fluorescence, independent of RNA degradation.

Students may additionally observe that the amount of green fluorescence varies over time across

the three different tubes. This is true even though all three tubes are producing mRNA.

For tube 2, fading of the green fluorescence may not be observed, as the green fluorescence fades

aroundthesametimeastheredfluorescencestartsbecomingvisible(threetofourhoursafterthe

startofthereaction),maskingthelossofgreenfluorescence.Studentsmaynoticeashiftfromgreen

fluorescencetoyellow/orangefluorescencebeforeobservingtheexpectedredfluorescence.Yellow/

orange fluorescence will occur when both the green fluorescent aptamer and the red fluorescent

protein are expressed simultaneously. Eventually, the green fluorescence from the aptamer will fade,

as the red fluorescent protein reaches its peak brightness, leaving only the red fluorescence visible

at around 8 hours.

For tube 3, fading of the green fluorescence is typically observed after three to four hours.

For tube 4, the green fluorescence may appear brighter than tubes 2 or 3 and the fading of the

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Expected Results

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green fluorescence is not observed until around hour five to six. This brighter, longer lasting green

fluorescence may be due to the fact that DNA B does not contain a protein coding sequence or

ribosomal binding site, thus extending the effective life of the RNA aptamer. Using this fact, you may

invite your students to offer plausible hypotheses that would explain this observation.

Observations concerning red fluorescence:

In tube 2, it typically takes approximately 8 hours for full red fluorescence to be observed. This may

seem longer than would typically be expected for most proteins to be expressed; theoretically, a

protein of the size made in this lab should be able to be fully translated and folded within just a few

minutes of the start of transcription. However, fluorescent proteins undergo a post-translational

process called maturation. Maturation involves the spontaneous rearrangement of amino acids at

thecenteroftheproteintoformthematurechromophore(thepartoftheproteinresponsiblefor

thefluorescentcolor).Dependingonthespecificfluorescentproteinandthesysteminwhichitis

expressed, this process can take as little as under ten minutes or as long as several hours. The red

fluorescent protein used in this lab has a long maturation time. This means that even though the first

mRNAs are likely translated within minutes, it takes several hours for enough fluorescent proteins to

complete maturation to be visible.

Optional extension questions

If you would like to explore these ideas and explanation with your students, here are optional

relevant questions that can be used to inspire classroom discussions.

1)Youmaynoticethatthegreenfluorescencebeginstofadeintubes2-4afterafewhoursand

is the same as the baseline negative control the next day. Propose an explanation of why you

believe this green fluorescence disappears.

2)Ifyouwereabletoobserveyourtubesafter3-4hours,youmayhavenoticedorangeor

yellow fluorescence in tube 2, even though no orange or yellow fluorescent molecules or

proteinsarepresentinthisreaction.Proposeanexplanationofwheretheorange/yellow

fluorescence came from and why they “disappeared” over time.

3)Ifyouwereabletoobserveyourtubesafter3-4hours,youmayhavenoticedthatwhile

tube 3 no longer had visible green fluorescence, tube 4 still had green fluorescence, which

wouldnothavefadeduntilaroundhour5-6.SincetheDNAusedintube4(DNAB)doesnot

containaproteincodingsequenceorribosomalbindingsite,theresultingmRNAwill1)be

shorterthanthemRNAresultingfromDNAAand2)notbindtotheribosome.Proposean

explanation of why DNA B may result in a longer visible green fluorescence than DNA A.

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Expected Results

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4)Whilegreenfluorescenceisvisiblewithinminutes,redfluorescenceisonlyvisibleafter

3-4 hours and does not reach full fluorescence until around hour 8. Research typical rates of

transcription and translation. Do the times observed here match typical protein expression

rates? Investigate the steps involved in the expression of fluorescent proteins specifically. Using

this information, can you propose an explanation for the fluorescence times you observed in

this reaction?

P./30

Student’s Guide

Overview P.31

Extension: RNA aptamers P.32

Study questions P.35

Additional

Student Supports

and Extension

Activities

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Additional

Supports

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Overview

The activities in this section are provided as optional activities to do in conjunction with this

BioBits

lab. These activities are not included in the Student’s Guide.

Additional supports

The following optional supports are to help provide additional scaffolding for students who need it.

Micropipetting 101 – Students performing this lab should be familiar with proper micropipette

technique and be able accurately to pipette volumes in the 2-5 μL range. This introductory pipetting

activity will introduce students to proper pipetting technique and have them practice pipetting a

variety of volumes. (http://www.minipcr.com/micropipetting/)

Alternatively, have students practice pipetting small volumes of food coloring beforehand.

Extension activities

This BioBits

labisbasedoncutting-edgefreeze-dried,cell-free(FD-CF)technology.Thiscentral

dogma lab is just one example of many uses that FD-CF has in education and in scientific research.

For students curious about the different applications of FD-CF technology, we encourage them to

read through these extension activities and answer the study questions to learn more about how

FD-CF reactions work and how they are used by scientists in real-world scenarios.

2. RNA aptamers: The green signal associated with RNA and

transcription in this lab is made possible with a structure called an RNA aptamer. If students are curious

about how RNA aptamers work and the role they played in the lab, this activity will have the students

read more about RNA aptamers and answer study questions about them.

3. Crick and the central dogma: Read more about about the history of Francis Crick’s lecture in this

60th anniversary article from PLoS Biology. Article is available for free download at the link below, or

searchusingthefollowingcitation:CobbM(2017)60yearsago,FrancisCrickchangedthelogicof

biology.PLoSBiol15(9):e2003243.https://doi.org/10.1371/journal.pbio.2003243

1. Cell-free protein synthesis: This BioBits

lab uses cell-free protein

synthesis technology to create a low-cost and easy-to-use hands-on

activity. Read more about how cell-free reactions work and its different

real-world applications. DNAdots include articles and study questions.

Browse free articles at: dnadots.minipcr.com

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Additional

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Extension: RNA aptamers

What they are and how they work

In this lab, BioBits

reactions use fluorescence as a tool to observe the production of both RNA and

protein. Without the use of these fluorescent markers, a BioBits

reaction would look like a clear

liquid before, during, and after transcription and translation. This is because DNA and RNA are trans

parent in solution and proteins frequently are as well. In this lab, however, we were able to observe

RNA as fluorescent green and protein as fluorescent red.

To visualize protein production, this lab used a red fluorescent protein. Fluorescent proteins come

inmanydifferentcolors(redandgreenaremostcommon),andarecommonlaboratorytoolsfor

visualizing cellular structures and processes. The fluorescent proteins used in laboratories are

derived from those found in nature, with modifications made by scientists to make these proteins

brighter and more stable. Through genetic engineering, scientists have adapted these naturally

occurring proteins for biotechnology uses.

To visualize RNA, this lab used an RNA aptamer. An aptamer is a sequence of DNA or RNA that

is capable of binding to a specific molecule. While there are some aptamers that occur in natural

systems, most aptamers used in biotechnology, unlike fluorescent proteins, are purely synthetic—

that is, they are designed from scratch by scientists for a specific purpose.

We normally think of DNA and RNA as long linear molecules. But in solution, these molecules

actually twist and fold to make complex three-dimensional structures. This is because the

nucleotidesthatmakeupDNA(A,T,C,andG)andRNA(A,U,C,andG)readilyformhydrogen

bonds and other electrostatic interactions when in solution. These nucleotides bind to each other

according to the rules of base pairing — C to G and A to T or U, which is why DNA forms a double

helix and how RNA is transcribed from DNA. But this binding potential also means that long RNA

molecules are typically more stable as twisted, folded structures, binding to themselves and even

other molecules for stability. If you are familiar with the structure of tRNAs or with the fact that the

ribosome contains both RNA and protein bound to each other, you are already familiar with this

phenomenon.

Synthetic biologists have taken advantage of this property of nucleic acids and have designed

specific nucleotide sequences that will fold into three-dimensional structures which then fit and bind

to a specific molecule of interest. They call these sequences aptamers from the Latin root aptus,

which means “to fit”.

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Additional

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Aptamersfunctionverysimilarlytoantibodies(proteinsusedbytheimmunesystemtospecifically

targetpathogens)andcanbeusedinmanyofthesamescenariosthatantibodiesareused.Thekey

difference is that antibodies are made of protein, while aptamers are comprised of nucleic acids. This

gives aptamers some unique properties. They can be added on to longer nucleic acid sequences

much like protein tags can be added to protein sequences. They also tend to be significantly smaller

than antibodies, and so in some cases, can better infiltrate tissues and cells for scientists to visual

ize these components. The bonds formed by aptamers tend to be weaker and more susceptible to

changes in temperature, which is the main reason why you incubated the BioBits

reactions at or

closeto37°Cwhenwaitingtoviewthegreenfluorescentsignal.RNAalsotendstobedegraded

relatively quickly in cells, and so RNA aptamers often have a much shorter lifespan once introduced

to the cellular environment.

In this lab, a sequence was added to the DNA so that when it is transcribed, the RNA will form an

aptamer. This aptamer binds to a fluorescent molecule very similar to the chromophore found in

greenfluorescentprotein(GFP).Attheheartofanyfluorescentproteinisthechromophore, a small

molecular structure that gives the protein its color. In the case of GFP, three amino acids in the

center of the protein are modified after translation to make the chromophore. The rest of the protein

structure stabilizes the chromophore and creates the correct molecular environment for it to

fluoresce.

Figure 1. The chromophore used in this

lab closely resembles the chromophore

found in GFP proteins.

Scientists have made a synthetic version of the GFP chromo-

phore(Figure1),anditisthismoleculethatisintheBioBits

pellet. The chromophore alone however is not fluorescent in

solution; to fluoresce, it requires a stabilizing structure that will

providethecorrectmolecularenvironment.(Thisiswhythe

BioBits

reaction does not fluoresce green immediately when

waterisadded.)Insteadofusingaprotein,scientistsdevel

oped an RNA aptamer to do just that. When this aptamer is

transcribed from DNA to RNA, it will fold up on itself and make

a specific three-dimensional structure that holds the chromo

phore in just the right way to allow it to fluoresce brightly.

Because the chromophore that this aptamer binds to will fluoresce green, the aptamer was given the

name Broccoli. By inserting the Broccoli sequence between the promoter and the start codon of a

coding sequence, when that particular mRNA is transcribed, the aptamer sequence will fold up and

bind to the chromophore if it is present. The rest of the RNA is unchanged and will function

normally. In the case of this lab it will go on to be translated by the ribosome, producing the red

fluorescent protein you observed. To make the RNA fluoresce even brighter, two broccoli aptamers

in a row are present in the RNA sequence.

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Additional

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The Broccoli aptamer was developed as a tool for scientists to image RNA in living cells. By adding

the Broccoli aptamer to a DNA sequence of interest, researchers can visually establish exactly when

that DNA sequence is transcribed, and even trace where in the cell the RNA goes. In this way, the use

of the Broccoli aptamer in BioBits

reactions mirrors the way researchers are using it in the lab, but

put in your hands using simple tools.

Figure 2. A single RNA strand folds into a double stranded structure. The circled regions will each bind to a chromophore in

solution.

Adaptedfrom:Filonov,G.S.,Moon,J.D,Svenson,N.andJaffrey,S.R.(1994).Broccoli:RapidSelectionofanRNAMimicofGreenFluorescentProteinby

Fluorescence-BasedSelectionandDirectedEvolution.JournaloftheAmericanChemicalSociety,136(46),pp.16299-16308.

miniPCR bio

Learning Lab. BioBits

: Central Dogma

Additional

Supports

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Study questions

1. How is an RNA aptamer similar to a protein? How is it different?

2. Why do you think it would be important for scientists to track the RNA in cells, instead of tracking

DNA?

3. If you looked at your freeze-dried BioBits

pellets(beforeaddinganyliquid)underthebluelight,

you would see the green signal. Why do you think this might be?

4. The green fluorescence from the RNA aptamer was likely gone when you viewed your sample the

next day. But the fluorescent protein that was made will last for weeks? Can you suggest why that

may be using information from the text?

5. RNA is generally considered a single-stranded molecule. How would you describe the structure in

Figure 2?

The sequence below is the aptamer sequence as it exists in the DNA that you added to your tubes.

Use the sequence and the diagram of the aptamer sequence from the text to answer the questions

below.

miniPCR bio

Learning Lab. BioBits

: Central Dogma

Additional

Supports

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6.WhichstrandoftheDNA(toporbottom)wastheaptamertranscribedoff.Inotherwords,which

strand does the RNA polymerase use as a template? Add a 5’ and 3’ label to each strand of DNA if

you are able.

7.FindthebroccoliunitsintheDNAtemplatesequence(thestrandyouindicatedinQuestion6).

Circle the parts of the sequence that are part of a broccoli unit. Label each circle either A or B to

indicate which Broccoli unit it belongs to.

8. This structure can be described as a stem-loop, also sometimes called a hairpin. Draw a vertical

line in your sequence indicating where the sequence folds over to bind to itself.

9. Is the line in your sequence in the exact middle of the sequence? If not, explain how the point

where the sequence folds to bind to itself is not in the middle. Mark on the sequence with an

asterisk(*)theregionsthatcausethisasymmetry.

Student’s Guide

P./37

Teacher’s notes and placement in unit P.38

Learning goals and skills developed P.39

Standards alignment P.40

Ordering information P.41

About miniPCR bio™ Learning Labs P.42

Teacher’s

Notes

miniPCR bio

Learning Lab. BioBits

: Central Dogma

miniPCR bio

Learning Lab. BioBits

: Central Dogma

P./38

Instructor’s Guide

Teacher’s notes and placement

in unit

Teacher’s notes

Beforeperformingthislabstudentsshouldhavethefollowingskills/backgroundknowledge:

• Basic competence using a micropipette

• Basic understanding of DNA, RNA and protein

• Understanding of proteins and their synthesis

See the additional supports section of this lab for ways to scaffold this assignment for students who

may be less comfortable with the above skills.

This lab is designed to be performed in classes ranging from introductory life science to advanced

biotechnology classes. For more introductory classes, focus most strongly on the order of colors

that you will observe and what those colors represent. For more advanced classes, dive deeper into

the molecular processes and specific molecules that are active in each step of the reaction, as well

the cell-free technology used in the activity.

Placement in unit

Transcription and translation

This lab is designed for students to experiment with transcription and translation in an authentic and

meaningful way. Use this lab to reinforce concepts at the end of your transcription and translation unit

or at the beginning of a unit as a way to introduce the concepts and important biological molecules

involved.

Biotechnology and synthetic biology

This lab uses cutting-edge freeze-dried cell-free technology. Use this lab to introduce students to

the field of synthetic biology, focusing on the potential uses of cell-free technology. As part of this

approach follow this lab up by reading more about cell-free technology in DNAdots™, a free resource

fromminiPCRbio™(dnadots.minipcr.com).Also,haveyourstudentslearnmoreabouttheaptamer

technology used in this lab in the included extension.

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Learning Lab. BioBits

: Central Dogma

P./39

Instructor’s Guide

Learning goals and skills developed

Student Learning Goals – students will:

• Distinguish between individual steps of the central dogma of molecular biology

• Describe the function of each step of the central dogma and their relationship to each other

• Predict how manipulating different steps in the central dogma will affect observations

• Analyze and construct an explanation based on the results of an unknown reaction

Scientific Inquiry Skills – students should be able to:

• Formulate hypotheses and predict results

• Compare results to their predictions and draw conclusions based on hypotheses

• Identify potential sources of experimental error and their impact

Molecular Biology Skills:

• Micropipetting

• Use of fluorescence to identify nucleic acids and proteins

• Cell-free protein synthesis

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Learning Lab. BioBits

: Central Dogma

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Instructor’s Guide

Standards alignment

Next Generation Science Standards alignment

Students who demonstrate understanding can:

HS-LS1-1.

Construct an explanation based on evidence for how the structure of DNA determines the structure

of proteins which carry out the essential functions of life through systems of specialized cells.

HS-LS3-1.

Ask questions to clarify relationships about the role of DNA and chromosomes in coding the instructions

for characteristic traits passed from parents to offspring.

Science and Engineering

Practice

Disciplinary Core Ideas Crosscutting Concepts

•AskingQuestionsandDening

Problems

• Developing and Using Models

• Planning and Carrying Out

Investigations

• Analyzing and Interpreting Data

• Constructing Explanations and

Designing Solutions

• Engaging in Argument from

Evidence

• Obtaining, Evaluating, and

Communicating Information

LS1.A: From Molecules to Organisms: Structures

and Processes

•All cells contain genetic information in the form of

DNA molecules. Genes are regions in the DNA that

contain the instructions that code for the formation

of proteins, which carry out most of the work of

cells.(HS-LS1-1)

LS3.A: Inheritance of Traits

• Each chromosome consists of a single very long

DNA molecule, and each gene on the chromosome

is a particular segment of that DNA. The instructions

for forming species’ characteristics are carried in

DNA. All cells in an organism have the same genetic

content,butthegenesused(expressed)bythecell

may be regulated in different ways. Not all DNA

codes for a protein; some segments of DNA are

involved in regulatory or structural functions, and

somehavenoas-yetknownfunction.(HS-LS3-1)

• Patterns

• Cause and Effect

•Scale,Proportion,AndQuantity

• Systems and System Models

• Energy and Matter

• Structure and Function

• Stability and Change

• Interdependence of Science,

Engineering, and Technology

•InuenceofEngineering,

Technology, and Science on

Society and the Natural World

Common Core ELA/Literacy Standards

RST.9-10.1 Citespecictextualevidencetosupportanalysisofscienceandtechnicaltexts,attendingtothe

precise details of explanations or descriptions.

RST.9-10.3 Follow precisely a complex multistep procedure when carrying out experiments, taking measurements,

orperformingtechnicaltasks,attendingtospecialcasesorexceptionsdenedinthetext.

RST.9-10.4 Determinethemeaningofsymbols,keyterms,andotherdomain-specicwordsandphrasesastheyare

usedinaspecicscienticortechnicalcontextrelevanttogrades9-10textsandtopics

RST.9-10.5 Analyze the structure of the relationships among concepts in a text, including relationships among key

terms(e.g.,force,friction,reactionforce,energy).

RST.9-10.9 Compareandcontrastndingspresentedinatexttothosefromothersources(includingtheirown

experiments),notingwhenthendingssupportorcontradictpreviousexplanationsoraccounts.

WHST.9-10.1 Writeargumentsfocusedondiscipline-speciccontent.

WHST.9-10.2 Writeinformative/explanatorytexts,includingthenarrationofhistoricalevents,scienticprocedures/

experiments, or technical processes.

WHST.9-10.9 Drawevidencefrominformationaltextstosupportanalysis,reection,andresearch.

*For simplicity, this activity has been aligned to high school NGSS and grades 9-10 Common Core standards. This lab is easily

aligned to other grade levels as well.

miniPCR bio

Learning Lab. BioBits

: Central Dogma

P./41

Instructor’s Guide

Ordering information

To request miniPCR bio

Learning Labs reagent kits, you can:

 Call(781)-990-8PCR

email us at orders@minipcr.com

visit www.minipcr.com

BioBits® Central Dogma kit (catalog no. KT-1910-01) contains the following reagents:

• 32 BioBits

pellets in 8-tube PCR strips

• DNA Samples

• Nuclease-Free Water

• Kanamycin

Materials are sufficient for 8 lab groups, or 32 students.

Allcomponentsshouldbestoredasindicatedinthestoragenotes(seebelow).

Storage notes

* Pellets are viable for up to six months from the date of receipt when properly stored in the freezer

(approx.-20°C).

* Once opened, store unused pellets in the freezer in an airtight bag with the supplied orange

desiccant card.

*DNAandkanamycincanbestoredforupto12monthswhenkeptinthefreezer(approx.-20°C).

*Iffreezerstorageisnotavailable,thereagentsmaybestoredinthefridge(approx.4°C).Pellets

are viable for up to three months from the date of receipt when stored in the fridge. DNA and

kanamycin can be stored for up to six months when kept in the fridge.

miniPCR bio

Learning Lab. BioBits

: Central Dogma

P./42

Instructor’s Guide

About miniPCR bio

Learning Labs

and the BioBits®

system

This Learning Lab was developed by the miniPCR bio

team in an effort to help more students

understand concepts in molecular biology and to gain hands-on experience in real biology and

biotechnology experimentation.

We believe, based on our direct involvement working in educational settings, that it is possible for

these experiences to have a real impact in students’ lives. Our goal is to increase everyone’s love of

DNA science, scientific inquiry, and STEM. We develop miniPCR bio

Learning Labs to help achieve

these goals, working closely with educators, students, academic researchers, and others committed

to science education.

The BioBits

cell-free system was developed after realizing that the advantages of freeze-dried,

cell-free technology, such as simplicity of use, minimal equipment needs, and low cost, would

be ideal for a classroom setting to illustrate biomolecular concepts to students and teach basic

laboratory techniques. Researchers have been using cell-free reactions in their laboratories for years,

and the BioBits

system now makes this cutting-edge technology accessible anywhere to anyone

interested in learning molecular biology as an excellent teaching tool to enhance biology education

both within and beyond the classroom.

The guiding premise for this lab is that a simple freeze-dried, cell-free-based experiment can visually

recapitulateafundamentalbiologicalconceptusingareal-lifebiotechnologyapplication(BioBits

)

and provide the right balance between intellectual engagement, inquiry, and discussion.

Starting on a modest scale working with Massachusetts public schools, miniPCR bio™ Learning

Labs have been well received, and their use is growing rapidly through academic and outreach

collaborations across the world.