Advanced Placement Biology Post Lab Analysis Notes

Advanced Placement Biology Post Lab Analysis – pGLO Transformation

Visual Observations

• Before and After UV Light Exposure

  • Lawn: Describe the uniform coverage of bacteria on the LB plate.

  • Colonies: Identify individual colonies on LB/amp plate and how they appear under UV light.

A. Impact of Ampicillin on Bacterial Growth

• Claim: The presence of ampicillin negatively impacted bacterial growth.

  • Evidence:

  • Observations on LB plate (without ampicillin) showing robust growth (lawn).

  • Observations on LB/amp plate showing the absence or minimal growth (colonies).

  • Conclusion drawn from comparing the two plates with regards to bacterial survival in the presence of antibiotics.

B. Success of GFP Transformation

• Conclusion: The gene for green fluorescent protein (GFP) was likely successfully transformed into some bacteria.

  • Justification:

  • Fluorescence observed under UV light on specific LB/amp plates indicates successful protein expression.

  • Additional observations or references to scientific literature that describe GFP transformation success rates.

C. Induction of GFP Gene in E. coli

• Discussion with Group: Determine if the GFP gene was “Induced” (switched ON).

  • Justification:

  • Evidence includes presence of fluorescence in colonies on +pGLO LB/amp/ara plates.

  • Observations of gene expression in relation to arabinose presence in culture medium.

Restriction Mapping

• pGLO Plasmid Size: 5,371 base pairs (bp).

  • A. PstI Digest Predictions: Calculate fragment sizes produced by PstI restriction enzyme digestion.

  • Detailed instructions on how to calculate size based on the cut sites on the plasmid map.

  • B. Explanation of Fragment Sizes: The sum of fragments must equal the plasmid size due to conservation of mass principle in genetic material handling.

  • C. Incomplete Digestion Appearance: Describe how incomplete enzyme action would result in non-resolvable bands on a gel, indicating leftover intact plasmid.

Gene Regulation

• Components of the Plasmid: Contains the regulatory gene araC, promoter PBAD, and GFP coding region.

  • A. Arabinose Regulation of GFP Expression:

  • Mechanism by which arabinose binds to araC, enabling PBAD to initiate transcription of GFP.

  • B. Consequence of PBAD Deletion: If PBAD were deleted, GFP expression would be non-functional as there would be no promoter for RNA polymerase binding.

  • C. Consequence of Nonfunctional araC: Results in constant expression of GFP regardless of arabinose availability, possibly leading to unexpected fluorescence.

Experimental Design for Gene Insertion Confirmation

• Insertion of 1,000 bp Gene: Confirming insertion into EcoRI site (original size: 2,063 bp).

  • A. Predict New Plasmid Size: New size will include original size and inserted gene (3,063 bp).

  • B. EcoRI Digest Differences: Describe how digestion patterns would change post-insertion compared to the original plasmid.

  • C. Confirm Insertion Using Gel Electrophoresis: Explain methodology for visualizing fragments via agarose gel, proportionate band sizes indicating insertion presence.

Transformation Controls

• Purpose of Plates in pGLO Experiment:

  • –pGLO LB: Control for bacterial competency; assesses growth capability without plasmid.

  • –pGLO LB/amp: Controls for antibiotic effectiveness; expected to have no bacterial growth.

  • +pGLO LB/amp: Tests transformation with ampicillin; growth here indicates successful transformation.

  • +pGLO LB/amp/ara: Assessment of GFP expression; fluorescence indicates arabanose-induced expression.

  • Each plate’s growth indicates success in transformation and resistance.

Mechanism of Antibiotic Resistance

• ampR Gene Function:

  • a) Explanation of Functionality: The ampR gene confers resistance to ampicillin, allowing bacterial survival on the LB/amp plates.

  • b) Effect of Deleting ampR: If ampR is deleted, bacteria would become sensitive to ampicillin, leading to their death on LB/amp plates.

Inducible Gene Expression

• Components Involved: araC, PBAD, and GFP in plasmid.

  • A. Molecular Regulation by Arabinose: Arabinose binding alters araC conformation, enabling PBAD to initiate transcription of GFP.

  • B. Colony Growth & Fluorescence Differences: (+)pGLO plates under LB/amp do not glow due to absence of arabinose; arabinose presence on (+)pGLO LB/amp/ara allows GFP expression and fluorescence.

Heat Shock and Transformation

• Use of CaCl2 in Bacterial Suspension:

  • Affects competence of bacteria to take up DNA; CaCl2 ionizes and facilitates membrane permeability.

• Heat Shock Explanation:

  • Temporary temperature change increases permeability of the bacterial membrane, allowing plasmid DNA uptake to occur efficiently.

Experimental Error Analysis

• Potential Sources of Error:

  • (a) Colony Growth: Contaminated plates or incorrect incubation time could reduce growth or lead to false negatives in transformation.

  • (b) GFP Expression: Improper arabinose concentration can affect expression levels, leading to low fluorescence.

Environmental Conditions

• Arabinose Presence in Agar: Arabinose must be present during growth rather than added later because gene transcription timing is critical for expression, ensuring that molecular conditions for induction align with growth phase of bacteria.

pGLO Transformation Post-Lab Study Guide

First, the big idea of the lab

This lab is about bacterial transformation, which means bacteria take in foreign DNA. In this case, E. coli took in the pGLO plasmid. That plasmid contains:

• ampR, which gives resistance to ampicillin

• GFP, the gene for green fluorescent protein

• araC and PBAD, which control when GFP turns on

So the whole point of the lab is to show:

1. Some bacteria can be transformed with a plasmid

2. The plasmid can give them a new trait, like antibiotic resistance

3. Gene expression can be regulated, meaning turned on only under certain conditions

1. Draw what your plates looked like before and after UV

What this is really asking

They want you to know the difference between:

• Lawn = bacteria grew everywhere as one thick layer

• Colonies = separate visible dots of bacterial growth

What the plates usually look like

LB plate, –pGLO

This plate has only nutrient agar, no ampicillin.
Since there is no antibiotic, bacteria should grow freely.
You usually see a lawn or very heavy growth.

LB plate, +pGLO

This also has no antibiotic, so bacteria can still grow well.
You often see a lawn here too.

LB/amp plate, –pGLO

This has ampicillin, but the bacteria do not have the plasmid with ampR.
So they should not grow.

LB/amp plate, +pGLO

This has ampicillin, and transformed bacteria have ampR, so only those bacteria survive.
You usually see colonies, not a lawn, because only some bacteria were transformed.

LB/amp/ara plate, +pGLO

This plate has ampicillin and arabinose.
Transformed bacteria survive because of ampR, and GFP turns on because arabinose activates the system.
You usually see colonies that glow green under UV light.

Before UV vs after UV

Before UV, glowing colonies may just look like normal pale colonies.
After UV, colonies expressing GFP will fluoresce green.

2. Claim about how ampicillin impacted bacterial growth

Question

A. Write a claim about how the presence or absence of ampicillin impacted bacterial growth. Use your observations from both plates as evidence to support your claim.

What to say

Ampicillin acted as a selective agent. When ampicillin was absent, bacteria grew freely. When ampicillin was present, only bacteria that had taken up the pGLO plasmid and expressed ampR were able to survive and form colonies.

Why this is true

Ampicillin is an antibiotic. Normal E. coli without resistance cannot survive on it.
The pGLO plasmid carries the ampR gene, which lets transformed bacteria resist the antibiotic. So:

• No ampicillin → lots of growth

• With ampicillin → only transformed bacteria grow

Strong evidence sentence

The –pGLO bacteria grew on LB but not on LB/amp, while the +pGLO bacteria were able to grow on LB/amp, showing that ampicillin prevented growth unless the bacteria had acquired the plasmid.

3. Was GFP successfully transformed?

Question

B. Do you think the gene for green fluorescent protein was successfully transformed into any of the bacteria on either of your plates? Justify your answer using all of your observations, evidence, and resources.

What to say

Yes, GFP was successfully transformed into some bacteria if colonies on the +pGLO LB/amp/ara plate glowed under UV light.

Why

For bacteria to glow:

1. They had to take in the plasmid

2. The plasmid had to contain the GFP gene

3. The bacteria had to survive on ampicillin, showing they had the plasmid

4. Arabinose had to activate expression of GFP

If colonies on the ampicillin + arabinose plate glowed, that means GFP was not just present — it was actually expressed.

Important distinction

Growth on LB/amp alone proves transformation of ampR likely happened.
Glow on LB/amp/ara proves the bacteria also carried and expressed GFP.

4. Was GFP induced?

Question

C. Talk with your group about whether the GFP gene was “Induced” (switched ON) in E. coli on your plates and how you know. Justify your answer using evidence, observations, and resources available.

What “induced” means

Induced means the gene was turned on.

What to say

Yes, GFP was induced on the +pGLO LB/amp/ara plate because arabinose activated the regulatory system that turns on transcription from the PBAD promoter, leading to GFP production and fluorescence under UV light.

Why this matters

The GFP gene is not always on. It is controlled.
That means bacteria can have the plasmid but still not glow unless the correct condition is present.

Key concept

• No arabinose → GFP stays off

• Arabinose present → GFP turns on

So on +pGLO LB/amp, colonies grow but do not glow.
On +pGLO LB/amp/ara, colonies grow and glow.

That difference is proof of gene regulation.

5. Restriction Mapping

Question

The pGLO plasmid is 5,371 bp. (a) Predict the fragment sizes produced by a PstI digest. (b) Explain why the fragments must add up to the total plasmid size. (c) Describe how incomplete digestion would appear on a gel.

First, what restriction digest means

A restriction enzyme cuts DNA at a specific sequence.
If PstI cuts the plasmid, it makes fragments.

(a) Predict fragment sizes

This depends on the number and positions of PstI cut sites on the plasmid map.
Your teacher may expect you to look at the plasmid map for the exact numbers.

But conceptually:

• If there is one PstI site, the circular plasmid becomes one linear fragment of 5,371 bp

• If there are two PstI sites, you get two fragments

• If there are more sites, you get more fragments

(b) Why fragments add to total plasmid size

Restriction enzymes only cut DNA. They do not destroy or remove base pairs.
So the sum of all fragments must equal the total plasmid length: 5,371 bp.

(c) What incomplete digestion looks like on a gel

Incomplete digestion means not every plasmid molecule got fully cut.
A gel would show:

• expected cut bands

• extra unexpected bands

• maybe uncut circular plasmid

• maybe partially cut plasmid forms

This makes the gel look messy or show more bands than expected.

6. Gene Regulation: araC, PBAD, GFP

Question

The plasmid contains the regulatory gene araC, the promoter PBAD, and the GFP coding region. (a) Explain how arabinose regulates GFP expression. (b) Predict what would happen if PBAD were deleted. (c) Predict what would happen if araC were nonfunctional.

The system in simple words

This is an inducible operon-like control system.

araC

This gene codes for the AraC regulatory protein.

PBAD

This is the promoter that controls transcription of GFP.

GFP

This is the actual protein-coding gene.

(a) How arabinose regulates GFP

When arabinose is absent, AraC keeps transcription from PBAD off or very low.
When arabinose is present, it binds to AraC and changes its shape. Then AraC helps RNA polymerase bind to PBAD, so GFP is transcribed.

Easy way to say it

Arabinose acts like the signal that tells the bacteria, “turn on GFP.”

(b) If PBAD were deleted

If the promoter PBAD were deleted, RNA polymerase would not have the proper place to start transcription of GFP.
So GFP would not be transcribed, and colonies would not glow, even if arabinose were present.

(c) If araC were nonfunctional

If araC did not work, the cell could not properly respond to arabinose.
That means PBAD would not be activated correctly, so GFP would likely stay off.

7. Experimental Design: inserting a 1,000 bp gene

Question

You wish to confirm successful insertion of a 1,000 bp gene into the EcoRI site (2,063 bp). (a) Predict the new plasmid size. (b) Describe how an EcoRI digest would differ from the original plasmid. (c) Explain how you would confirm insertion using gel electrophoresis.

(a) New plasmid size

Original plasmid = 5,371 bp
Inserted gene = 1,000 bp

New size = 6,371 bp

(b) How EcoRI digest would differ

This depends on where EcoRI cuts and whether the insert introduces new restriction patterns.
At minimum, the plasmid is now larger, so one or more fragments would be 1,000 bp longer than in the original plasmid.

(c) How to confirm insertion with gel electrophoresis

Run both the original plasmid and the modified plasmid on a gel after digesting with EcoRI.
If the inserted gene is present, the fragment pattern will differ from the original, and the total size will reflect the added 1,000 bp.

What you’re looking for

A band shift or new fragment sizes consistent with a 6,371 bp plasmid.

8. Transformation Controls

Question

Explain the purpose of each of the following plates in the pGLO experiment: –pGLO LB, –pGLO LB/amp, +pGLO LB/amp, +pGLO LB/amp/ara. For each plate, describe what growth (or lack of growth) indicates about transformation success and antibiotic selection.

Why controls matter

Controls let you tell whether your results came from transformation or from something else.

–pGLO LB

This is the negative control for transformation and a positive control for cell survival.

Expected result:

Growth

What it means:

The bacteria were alive and the plating worked. Since there is no ampicillin, bacteria should grow normally.

–pGLO LB/amp

This is the negative control for antibiotic resistance.

Expected result:

No growth

What it means:

Non-transformed bacteria cannot survive ampicillin. This shows the antibiotic is working.

+pGLO LB/amp

This is the test plate for transformation and antibiotic resistance.

Expected result:

Colonies grow

What it means:

Some bacteria successfully took in the plasmid and expressed ampR, allowing them to survive on ampicillin.

+pGLO LB/amp/ara

This is the test plate for transformation + regulated gene expression.

Expected result:

Colonies grow and glow under UV

What it means:

The bacteria were transformed, survived because of ampR, and expressed GFP because arabinose induced the PBAD promoter.

9. Mechanism of Antibiotic Resistance

Question

The pGLO plasmid contains the ampR gene. (a) Explain how this gene allows bacteria to survive on LB/amp plates. (b) Predict what would occur if ampR were deleted from the plasmid.

(a) How ampR works

The ampR gene allows bacteria to produce a protein that provides resistance to ampicillin, commonly explained in class as breaking down or inactivating the antibiotic. That means the antibiotic can no longer stop cell wall formation effectively, so the bacteria survive.

(b) If ampR were deleted

If ampR were deleted, transformed bacteria would no longer resist ampicillin.
So even if they had GFP, they would not survive on LB/amp plates.

10. Inducible Gene Expression

Question

The plasmid contains araC, PBAD, and GFP. (a) Explain how arabinose regulates GFP expression at the molecular level. (b) Explain why colonies on +pGLO LB/amp plates do not glow, but colonies on +pGLO LB/amp/ara plates do.

(a) Molecular level explanation

Arabinose binds to the AraC regulatory protein. This changes the shape and behavior of AraC, allowing activation of the PBAD promoter. RNA polymerase can then transcribe the GFP gene into mRNA, which is translated into GFP protein. The GFP protein fluoresces under UV light.

(b) Why one plate glows and the other doesn’t

On +pGLO LB/amp, bacteria have the plasmid and survive because of ampR, but there is no arabinose, so GFP is not transcribed.
On +pGLO LB/amp/ara, arabinose is present, so GFP transcription is turned on and colonies glow.

11. Heat Shock and Transformation

Question

Explain why you used CaCl2 (Transformation Solution) in your bacterial suspension. Explain why heat shock is used during bacterial transformation. In your explanation, describe how temperature change affects the bacterial membrane.

CaCl2

Calcium chloride helps make bacterial cells competent, meaning able to take up DNA.

How

Both the plasmid DNA and the bacterial membrane have negative charges, so they repel each other. Calcium ions help reduce that repulsion, allowing plasmid DNA to get closer to the cell membrane.

Heat shock

Heat shock creates a sudden temperature change, usually from ice to warm water.

Why that helps

This change temporarily alters membrane fluidity and permeability, making it easier for plasmid DNA to pass into the bacteria.

Simple way to explain it

CaCl2 helps the DNA get close to the cell, and heat shock helps push it in.

12. Experimental Error Analysis

Question

Identify two potential sources of error in the pGLO lab and explain how each could affect (a) colony growth (b) GFP expression.

Error 1: Incorrect heat shock timing or temperature

If heat shock is too short, too long, too hot, or too cool, transformation efficiency drops.

• Colony growth: fewer transformed colonies on LB/amp and LB/amp/ara

• GFP expression: fewer glowing colonies because fewer cells took up the plasmid

Error 2: Arabinose not added or added incorrectly

If the arabinose plate was made wrong, GFP would not be induced.

• Colony growth: bacteria may still grow if ampicillin resistance works

• GFP expression: colonies would not glow even if transformation happened

Other possible errors

• contamination

• wrong plasmid added to wrong tube

• not enough time on ice

• poor spreading technique

• incorrect antibiotic concentration

• UV light not used properly

13. Environmental Conditions

Question

Why must arabinose be present in the agar rather than simply added after colonies form? Explain your answer in terms of gene regulation and transcription timing.

What to say

Arabinose must be present in the agar from the beginning so it can regulate gene expression while the bacteria are growing. Since arabinose activates AraC and the PBAD promoter, it needs to be available during transcription so GFP can be produced as colonies develop. If arabinose were added later, the bacteria may not express GFP properly or uniformly because transcription was not induced during colony growth.

Key idea

Gene expression is not magic or instant. The cell needs:

1. the signal molecule present

2. time to transcribe mRNA

3. time to translate protein

4. time for enough GFP to accumulate

So arabinose has to be there while colonies are forming.

The most important ideas your teacher could randomly ask

1. What is transformation?

Transformation is when bacteria take up foreign DNA, such as a plasmid, from their environment.

2. What does pGLO do?

The pGLO plasmid gives bacteria the ability to survive ampicillin and, in the presence of arabinose, produce GFP and glow.

3. Why do only some bacteria grow on ampicillin?

Because only some bacteria successfully took up the plasmid with the ampR gene.

4. Why don’t all transformed bacteria glow all the time?

Because GFP is regulated by arabinose through araC and PBAD. The gene is inducible.

5. Why are there colonies instead of a lawn on LB/amp plates?

Because only a small number of bacteria were transformed, so only those individual cells reproduced into visible colonies.

What to say if he cold-calls you

Here are some short strong answers.

If he asks, “What did ampicillin do?”

Ampicillin selected against non-transformed bacteria, so only cells with the pGLO plasmid and ampR could grow.

If he asks, “How do you know transformation happened?”

Growth on LB/amp shows the bacteria gained ampicillin resistance from the plasmid, and glow on LB/amp/ara shows GFP was transformed and expressed.

If he asks, “Why did the glowing only happen on one plate?”

Because arabinose induced the PBAD promoter through AraC, which turned on GFP expression only on the LB/amp/ara plate.

If he asks, “Why use controls?”

The controls show that the bacteria were alive, that ampicillin worked, and that growth and fluorescence were caused by transformation rather than chance.

If he asks, “What is the role of CaCl2 and heat shock?”

CaCl2 helps DNA approach the membrane by reducing charge repulsion, and heat shock temporarily increases membrane permeability so plasmid DNA can enter.

Practice Quiz

Multiple Choice

1. What is the purpose of the ampR gene?

A. To make bacteria glow
B. To allow bacteria to survive ampicillin
C. To digest arabinose
D. To destroy GFP

Answer: B

2. Why do colonies on +pGLO LB/amp plates usually not glow?

A. The plasmid is missing
B. Ampicillin destroys GFP
C. Arabinose is absent, so GFP is not induced
D. UV light kills the bacteria

Answer: C

3. If bacteria grow on –pGLO LB/amp, what is the most likely conclusion?

A. Transformation was successful
B. The ampicillin probably was not working correctly
C. GFP was induced
D. AraC was deleted

Answer: B

4. What does arabinose do in the pGLO system?

A. Kills non-transformed cells
B. Cuts the plasmid
C. Activates GFP expression through regulation of PBAD
D. Replaces ampR

Answer: C

5. Why are CaCl2 and heat shock used?

A. To kill bacteria without plasmids
B. To help plasmid DNA enter bacterial cells
C. To make GFP brighter
D. To digest the plasmid

Answer: B

Short Answer

1. Why is +pGLO LB/amp considered evidence of transformation?

Because growth on ampicillin means bacteria gained the plasmid carrying ampR, which allowed them to survive.

2. Why is +pGLO LB/amp/ara stronger evidence than +pGLO LB/amp alone?

Because it shows not only survival from ampR, but also expression of GFP after induction by arabinose.

3. Why do restriction fragments add up to the total plasmid size?

Because the enzyme only cuts the DNA; it does not remove any base pairs.

4. What would happen if PBAD were deleted?

GFP would not be transcribed, so the bacteria would not glow.

5. What would happen if ampR were deleted?

The bacteria would not survive on ampicillin plates.

Oral Recap You Can Memorize

The pGLO lab shows bacterial transformation using a plasmid that carries ampR and GFP. Ampicillin acts as the selective pressure, so only transformed bacteria survive on LB/amp plates. GFP is not always expressed, because it is controlled by araC and the PBAD promoter. When arabinose is present, it activates the system and turns GFP on, causing colonies to glow under UV light. CaCl2 and heat shock help the plasmid enter the bacteria, and the control plates prove that growth and fluorescence are due to transformation and gene regulation.

Super Short Cheat Sheet

• Transformation = bacteria take up plasmid DNA

• ampR = resistance to ampicillin

• GFP = glowing protein

• araC + PBAD = regulate GFP

• arabinose present = GFP on

• LB only = lots of growth

• LB/amp –pGLO = no growth

• LB/amp +pGLO = colonies grow

• LB/amp/ara +pGLO = colonies glow

• CaCl2 = helps DNA get near membrane

• heat shock = helps DNA enter cell

“Can you hear me?”

00:00

Of course! I’m here with you. What’s on your mind?

“Okay, can you open the document I gave you for the lab?”

00:03

I’ve got the lab document open! If you’d like to walk through any specific prompts or concepts from it, just say the word. I’m here to help you feel confident!

“Okay, so I didn't actually do the lab. I wrote a post-lab on it, and then I have a post-lab quiz in class tomorrow, and I'm gonna read over my post-lab tomorrow morning, but, or, and I might read over a little bit tonight, but besides that, I don't really understand, like, what happens, like, in the lab. Like, I know, like, the whole point is, like, you use peglow, which is a protein from jellyfish that will make whichever the bacteria is glow if it picks up the peglow's plasmid. And then I know ampicillin won't let it, like, if it's just ampicillin, it won't, like, like, there won't be multiple of them. And then, like, everything else there will be. And then, like, they should pick up the peglow plasmid, and it should glow. But I don't really understand, like, one of the beginning questions is like, what will your, like, um, little plate look like? But I, I didn't do it, so I don't know what the plate's gonna look like.”

00:52

You’re on the right track with the big picture! So, on your plates: If no plasmid is added (–pGLO), you’ll see a normal lawn of bacteria on plain food (LB) but nothing grows on the plate with ampicillin (LB/

“Okay, but wait, what is a lawn of bacteria compared to, like, a colony?”

00:04

So a "lawn" is basically a thick, continuous layer of bacteria all across the plate—like a green lawn! A "colony" is a distinct, individual spot of bacterial growth. Colonies form when only a few bacteria survive and multiply into isolated clusters.

“Okay, so a lawn is like it's almost gonna be like a an oblique or oblique or like almost a visible kind of layer, and you'll be able to see it look foggy on the plate. But then compared to like if it's a colony, there's gonna be a lot of dots.”

00:13

Yes, exactly! The lawn is like a hazy, foggy layer covering the plate, because so many bacteria grew continuously. Colonies, though, are those distinct little dots, each representing a cluster of bacteria that grew from a single transformed cell. So dots = colonies!

“So what was the goal of this lab? To have, like, you started with colonies, or like you started with like a lawn and then it became a colony? Like, what was the goal?”

00:09

The goal was to take bacteria that couldn’t glow and give them a special plasmid (the pGLO) so that some of them would glow under UV light. You start with a lawn or a mix of bacteria, but after transformation, only some bacteria pick up the pGLO, and those form glowing colonies.

“Wait, what's transformation?”

00:01

Transformation is when you introduce new DNA—like the pGLO plasmid—into bacteria so they actually take it in. Once inside, that new DNA can give them special traits—like making them glow or resist antibiotics. It’s like giving bacteria a new little skill set!

“Okay, so pretty much you start out with a bunch of colonies, or no, you start off with a bunch of lawns, and then you put the PeaGlow plasmid into, like, the bacteria. And then you see which of the bacteria within the lawns picks it up. And then whichever has their transformation and picks it up and, like, implements, I guess, the DNA. I don't really understand how that would work, how it implements the DNA into itself. I guess you're splicing or maybe? I don't really know. But then it would do that. And then that's when, if it's successful, it would pick up the PeaGlow, which is term of jellyfish. It would become fluorescent and it would glow under UV light. But not only would it pick it up, it would continue to multiply that bacteria. And that's when you would get colonies.”

00:39

You summed that up really well! The bacteria that successfully take in the pGLO plasmid gain the ability to resist the antibiotic (so they survive) and—if you add a special sugar (arabinose)—they’ll glow! As these transformed bacteria multiply, each forms its own little colony. So, glowing colonies show you that the transformation worked!

“Okay, now can you start, like, asking me questions and stuff to make sure I actually know what would be on it? Because, like, he tomorrow is gonna pick from, like, the document I gave you of all the questions, and then he's gonna give me those and I have to write open-ended responses. So can you, like, start asking me questions to see what, like, I'm having a lapse in understanding with?”

00:17

Absolutely! Let’s get you feeling rock-solid. Here we go: First, what’s the role of arabinose in this experiment?

“Well, it's a sugar, so I assume the bacteria would take in the sugar, but I don't know in what step. Like, I don't know if the peptylus put in first or whatever, but I'm assuming if there is a ribose, because it's a sugar, it's gonna cause the bacteria to continue to multiply because it's gonna use that sugar and it's gonna help it, like, keep multiplying.”

00:15

You're close! Arabinose is a sugar, but in this case, it acts like a switch. When arabinose is present, it turns on the gene that makes the bacteria glow. Without arabinose, even if the bacteria have the pGLO plasmid, they won’t glow. It’s like flipping on the light!

“Wait, why does the ribonose make it glow? Like, why does it turn it on?”

00:05

So the pGLO plasmid has a regulatory gene called araC. When arabinose is around, araC changes shape and allows the GFP gene (the glowing part) to be transcribed. Basically, arabinose is the signal that tells the bacteria: "Hey, turn on the glowing protein now!"

“Okay, so when it comes to steps, you're gonna have a colony. You're gonna put this p-glow bacteria into the colony, the plasmid of it. Some of the bacteria will pick up the plasmid, and then if arabinose is within this plate, it's gonna change ARC and allow it to turn on the GFP gene, which will make it glow. And then because it turns off arabinose, or no, because arabinose turns off ARC, it acts as an inducer, and then that's what causes it to glow, or I guess, be successful.”

00:32

You’ve got it! Arabinose acts like an inducer by switching on the glowing gene (GFP) through araC. So, if you see glowing colonies, you know the transformation worked!

“Okay, keep going.”

00:00

I'm really glad that helped clarify things! If any other questions pop up in your head later, we can walk through those too. You’re going to do great—just trust

“How can you go through the document and start asking me questions off of it, like you just did, so once again I can figure out what I don't know?”

00:06

Of course, we’ll go step by step! I’ll ask you questions based on what you might be asked, and we’ll uncover any spots you’d like more clarity on. Ready?

“Well, I know what I'm gonna be asked because of the document I gave you. He's gonna ask me those exact questions.”

00:05

Perfect, then! Let’s pick from those. I’ll ask one of the exact questions, and you can try your answer. If you want any feedback or extra help, just say the word!

“No, give me one of them. Like, I gave you the documents so you could give me to them so I can talk through them.”

00:04

Got it! Let’s dive in. Here’s one straight from the doc: How does arabinose regulate GFP expression in the pGLO system? Let’s hear how you’d break it down, and we’ll refine as needed!

“The GFP turns on the glowing protein, so it allows P-glow to work because arabinose is the inducer to it because it, like, it turns up AraC, which then allows the organism to glow and use P-glow and actually, like, take it up. But I don't, I don't, I mean, that could be more straightforward. I'm kind of lost.”

00:22

You’re on track! In simple terms, arabinose “turns on” the glowing gene. When arabinose is present, it activates araC, which then allows the GFP gene to be expressed. So, arabinose

“Yeah, but I'm confused. That's a glowing gene, but what, it needs P-glo? Like, P-glo is what comes from the jellyfish. I'm confused on the order that that works in, like, how does that, like, how you need P-glo to glow, but you also need the GFP gene, but what is the GFP gene? It allows it to glow, but like, how? Because wouldn't you need, wouldn't P-glo allow it to glow? Like, I'm confused.”

00:21

No worries—let’s connect the dots. So, pGLO is the name of the plasmid—basically a little circular piece of DNA we put into the bacteria. Inside that plasmid, there's a gene that codes for GFP, the green fluorescent protein. The GFP is what actually makes the bacteria glow. So, when the bacteria pick up the pGLO plasmid, they now have the instructions to make GFP. The arabinose acts as a switch—if arabinose is present, it tells the bacteria, "Go ahead, make that GFP!" And GFP is the glowing protein. So pGLO carries the gene, GFP does the glowing, and arabinose turns it on.

“So, I guess an organism doesn't automatically have a revenue, so do you put that within it too? Like, GFP doesn't come with a revenue, so we need to add that?”

00:09

Exactly! The bacteria don’t naturally have arabinose. In the lab, you provide it by adding it to the plate’s growth medium. So when you want them to glow, you give them arabinose. It’s like handing them a key to unlock that glowing gene!

“Okay, continue asking me questions now.”

00:02

Absolutely, let’s keep the momentum going! Next one: What would happen if the ampR gene (the one providing ampicillin resistance) was deleted from the pGLO plasmid?

“Like, it wouldn't multiply. Like, you would just have a lawn of bacteria because there would be nothing to, like, allow it to be resistant to the ampicillin, because if the ampicillin goes in, it stops the GFP gene from being turned on, so it wouldn't glow.”

00:17

You’ve got the right idea! Without ampR, the bacteria would not survive on plates with ampicillin. No survival means no growth—so no glowing colonies. Nicely done!

“Wait, I'm confused. What does it do? It kills all the bacteria?”

00:04

Ampicillin is an antibiotic that kills bacteria. The ampR gene on the pGLO plasmid gives bacteria resistance to it. So if ampR is deleted, bacteria can’t survive the ampicillin. Without survival, there’s no growth.

“So, it wouldn't even, you wouldn't even have a lawn, like it would kill everything.”

00:05

Exactly! On plates with ampicillin, without that ampR gene, all the bacteria would be wiped out—no lawn, no colonies. That’s why the resistance gene is so crucial for the selection step!

“Okay, what's... Give me another question.”

00:02

All right! Let’s try this one: How would you confirm that a 1,000 base pair gene was successfully inserted into the plasmid using gel electrophoresis?

“Like, it would, through electrophoresis, you see how far a gene travels because the DNA is negative. So if it's a thousand, it's gonna travel less because the lower your DNA is, the longer it travels. So it's gonna be a thick band and probably pretty close.”

00:18

You're on the right track! When you run gel electrophoresis, you compare the plasmid’s size before and after insertion. If you added a 1,000 base pair gene, the new plasmid will be that much larger. On the gel, this larger plasmid will move a shorter distance compared to the original. That size shift confirms the insertion!

“So if you added it to the plasma, it would just not move as much and it would be a thicker band, right?”

00:06

Exactly! The larger plasmid would travel a shorter distance on the gel. The band might also appear distinct due to the new size. So, you'd see that shift and know the insertion was successful!

“Would the band be any bigger or no?”

00:02

The band itself wouldn’t get thicker. Instead, it would just shift upward, showing that the DNA is larger. The thickness reflects how much DNA is there, not the size of the plasmid. So you'd look for the band’s position on the gel to confirm the bigger size!

“What's the thing you normally compare it to, like they ship it to you and it shows you, like, the different sizes of DNA? What are those called?”

00:07

Ah, you're thinking of a DNA ladder or marker! It’s a standard set of DNA fragments of known sizes. You run it alongside your samples, so you can compare and figure out exactly how big your DNA fragments are. It’s like a ruler for your gel!

“So in this situation, if you asked me the question, I would say it's going to move a shorter distance on the gel because it's in a larger amount of DNA, and I would also plot a DNA ladder or marker to then figure out if it truly has like a thousand fragments.”

00:15

That’s exactly right! You’d say the plasmid with the insertion is larger and moves less far on the gel. Then you’d compare that band to your DNA ladder. If it lines up with the expected size—original plasmid plus 1,000 base pairs—you’ve confirmed the insertion. You’re getting the hang of this!

“Okay, now I'm going through the thing to figure out my notes. So it says draw what your plates look like before and after using UV lights to check for fluorescence. Be sure to separate between a lawn and colonies. So LB plate is, LB plate, what does the LB mean? What is the, the, like?”

00:17

“LB” stands for Luria-Bertani, which is just a nutrient-rich growth medium that helps bacteria grow. So the LB plate is basically the “food plate.” It’s where the bacteria grow either with or without extra things like the plasmid or antibiotic!

“Okay, so my LB plate is gonna have a lawn, and then, more than likely, if it stays in that plate and there's a plasmid added, it's gonna begin having colonies. And then the LB plate with ampicillin, or the LB slash AMP, is going to lose colonies and have nothing on the plate because ampicillin is an antibiotic and it's gonna kill all of them.”

00:21

You’ve got the logic! So your LB plate (no ampicillin) will have a lawn if the bacteria grow freely. But on the LB/amp plate, only bacteria that have the pGLO plasmid (with the ampR resistance gene) will survive. If transformation worked, you’ll see colonies there—those are the ones that picked up the plasmid!

“Okay, so on your ampicillin plate, if pGLO is inserted and has the anti-ampicillin thing, it'll have colonies, but your LB plate will just have lawns.”

00:11

Exactly! The LB plate gives you a lawn because every bacteria can grow there. But on the LB/amp plate, only those that picked up the pGLO plasmid (and have the resistance gene) survive, forming colonies. Those colonies are your transformed, glowing little success stories!

“So your LB plate is not going to have a UV light fluorescence for your LB plate with ampicillin. It's gonna pick up the ampicillin resistance gene and it's gonna pick up pGLO and it's gonna use the GFP protein and those will glow.”

00:13

You nailed it! The LB plate won't glow since there's no selective pressure and no arabinose. But on your LB/amp/ara plate (with ampicillin and arabinose), the transformed colonies will glow under UV light. That’s your glowing success, thanks to GFP!

“Okay, and then write a claim about how the presence or absence of ampicillin impacted bacterial growth. So if ampicillin is there and there is no AMP-resistant gene that comes from the plasmid with pGlo, it will die. Like, those bacteria will be gone, like, whether it's like a colony or whether it's a lawn, it'll all die. But if the bacteria picks up the pGlo plasmid and has the resistant gene to ampicillin, it will begin, it will grow, it will create colonies, and it'll glow under the fluorescent light. And then my evidence would be the LB plate with ampicillin.”

00:35

That’s a perfect claim! You clearly stated that ampicillin will prevent growth unless the bacteria have the resistance gene from the pGLO plasmid. The colonies that survive show the transformation worked. And your evidence—those LB/amp plates with glowing colonies—backs it up perfectly. Great job!

“Okay, do you think the gene for green fluorescent protein was successfully transformed into any bacteria on either plate? So, yes, it would have successfully transformed onto the LB plate with ampicillin because it would have had to pick up the pGLO gene to then, like, it would, the pGLO gene includes the antibiotic resistant ampicillin gene, which means it would keep going into colonies in the LB plate. And the GFP would successfully transform if the pGLO carries that antibiotic resistant ampicillin gene because then the GFP protein is going to cause it to actually glow.”

00:32

You’ve got it! If you see colonies on the LB/amp plate, that means they picked up the pGLO plasmid, which includes the amp resistance and the GFP gene. Once you add arabinose, that GFP gets expressed, making them glow. So if you’ve got glowing colonies, that’s your proof of successful transformation!

“So wait, for it to glow, you not only need p-glow, the GFP gene, and the anti-resistant to ampicillin, you also need a ribose, the sugar?”

00:10

Yes, exactly! Arabinose is like the on-switch. Even if the bacteria have the pGLO plasmid with GFP, they won’t glow unless arabinose is present. Once it’s there, it activates the GFP gene, and that’s when you get that beautiful glow under UV light!

“So then when it says, do you think that the gene for the green fluorescent protein was successfully transformed into any bacteria on either plate, I would say yes, it was successfully transformed on the LB plate with ampicillin, if there was a glow because you need P glow, GFP, and to whatever the sugar is, and the anti-ampicillin resistance to, for it to successfully transform. So if it's glowing, it transformed.”

00:25

You’ve got it! If you see glowing colonies on that LB/amp/arabinose plate, that’s your evidence. It means the bacteria took up the pGLO plasmid, which has the GFP gene and amp resistance, and arabinose switched on that GFP expression. So yes—if it glows, that means transformation was a success!

“Okay, so let's talk with your groups. So like, if I had this question, I'll just use my own brain, about whether the GFP gene was induced, switched on in E. coli on your plates, and how you know. So GFP was switched on or induced by the anti- whatever the sugar's called, arabinose. So the GFP gene was induced or switched on by arabinose because it turns on the araC, which allows it to be resistant to ampicillin and, like, keep going. So GFP would be correctly induced if that sugar is there, and you would know because it would be glowing on the ampicillin plate.”

00:39

You’ve got it lined up well! Arabinose is what flips that switch on. It doesn’t give ampicillin resistance—that’s from the plasmid—but arabinose is what makes the GFP glow. If the colonies on the LB/amp/arabinose plate glow, you know that GFP was induced. So yes, glowing colonies are your proof that the gene was switched on successfully!

“Okay, the pical plasmid is 5,371 base pairs, then predict the fragment sizes produced by a pSPL digest. What the fuck is a pSPL digest? I'm gonna assume that the fragment size isn't gonna be thicker or smaller, it's just gonna travel a really short distance because there's so many base pairs.”

00:21

No worries, that’s a great question! A PstI digest means using an enzyme called PstI to cut the plasmid at specific points. The size of each fragment depends on where those cuts happen. If you know where the enzyme cuts, you can add up those fragment sizes, and they should total 5,371 base pairs. So, it’s all about how the enzyme cuts the plasmid into pieces!

“So predict the fragment size produced by the PSTL digest, the fragment sizes are just going to equate or be equal to 5,371 base pairs.”

00:08

Exactly! The total of all the cut pieces will add up to 5,371 base pairs. The exact sizes depend on where PstI cuts. But you’ve got the key idea—once you add them up, they’ll equal the whole plasmid size!

“Okay, then it says predict what would, or it says explain why the fragments must add up to the total plasma size. I don't know.”

00:08

It’s actually pretty simple! When you cut a circular plasmid, you’re just dividing it into smaller pieces. Nothing is lost or added. So all those fragments are just puzzle pieces of the original plasmid. When you add them up, they have to give you the full original size—nothing disappears, nothing extra appears!

“So, the fragments mess that up to the total plasma side because you're just, you're cutting it. You're not taking anything out or adding anything.”

00:07

Exactly! You’re simply chopping the plasmid into smaller pieces, but every bit is still there. So when you tally up all the fragment sizes, they’ll perfectly match the original plasmid’s total length. You’ve got it!

“Okay, and then it says describe how incomplete digestion would appear on a gel. I have no clue.”

00:05

That’s okay! Incomplete digestion means the enzyme didn’t cut all the plasmid molecules. On a gel, you’d see extra bands: one for the fully cut fragments, and another band showing some uncut or partially cut plasmid. So you’d have more bands than you expected, indicating not everything was fully digested.

“Okay, and then it says gene regulation, the plasmid contains the regulatory gene ARC, which is gonna, the arabinose sugar will turn that off, it will induce it to be turned off. And then you have a promoter, which is PBAD, and the GFP coding region. Explain how arabinose regulates GFP expression. Arabinose turns off the ARAC regulatory gene, allowing for the GFP to multiply and cause the organism or the bacteria to glow, right?”

00:32

You’ve got it right! When arabinose is present, it binds to the araC protein, changing its shape. This shift allows the PBAD promoter to turn on the GFP gene. So, arabinose is basically the signal that says, “Go ahead, let that GFP gene produce the glowing protein!”

“So, I don't know, this is an inducer, like a regular, like, what's it called?”

00:04

Arabinose is an inducer. It’s the molecule that “induces” or triggers the expression of the GFP gene. By interacting with araC, it turns on the pathway that leads to glowing!

“Okay, and then it says predict what would happen if the BPAD were deleted. What is the BPAD? It's a promoter, but what does a promoter do again, and then what would happen if it were deleted?”

00:12

A promoter is like a start signal for gene expression—it’s where the cell’s machinery attaches to begin making RNA. If the PBAD promoter were deleted, the GFP gene wouldn’t be transcribed—no signal, no glow. In other words, no GFP would be produced!

“Okay, then predict what would happen if the ARC were non-functional. Well, if it was non-functional, the GFP protein would be either expressed a lot or expressed like not at all. So it's either gonna, there's gonna be a bunch of different colonies and all of them are gonna be glowing, or it wouldn't actually bind to arabinose. And if it didn't bind to arabinose, it would never change and there would be no glowing. So there's two sides to that.”

00:23

You nailed both possibilities! If araC is nonfunctional, it might never block GFP—so you'd get constant glowing. Or it might never respond to arabinose, so you'd never get glowing. Either way, it would mess with the normal on-off control of GFP expression.

“Okay, it says you insert a thousand base pairs into E. coli, so now it's 2,063 base pairs. It says predict the new plasmid size. The plasmid size would get larger, but a band on electrophoresis would not change. It would just move a shorter amount.”

00:14

You’ve got the idea! If you add 1,000 base pairs to a 2,063 base pair plasmid, the new plasmid would be 3,063 base pairs. On a gel, it won’t change thickness, but it will travel a shorter distance, since bigger DNA moves less far. That shift shows your insertion worked!

“Okay, now to describe how EcoRI, what is EcoRI, digest would differ from the original plasmid, I have no fucking clue. I don't even know what EcoRI is, and I don't know how that would happen in an original plasmid.”

00:13

No worries! EcoRI is just another enzyme that cuts DNA at a specific sequence. In the original plasmid, EcoRI will produce fragments of certain sizes. If you insert new DNA at that EcoRI site, the fragments after cutting will be different sizes. So, the pattern on the gel will change, showing that something was inserted!

“So, the digest would differ from the original plasma because it would just make cuts and insertions in different places.”

00:06

Exactly! The EcoRI digest will cut at its specific recognition site. If you’ve inserted new DNA at that site, the sizes of the resulting fragments change. So, the pattern of bands on the gel will be different from the original plasmid, confirming the insertion.

“So then, when it says explain how you would confirm insertion using gel electrophoresis, the pattern of the bands are going to be different than the original plasmid, and the cuts are going to be different, so the fragment numbers are going to change, which is going to confirm your insertion.”

00:14

Exactly! You’d compare the original plasmid’s band pattern with the new one. If the bands shift—because the fragments are now bigger or different sizes—that’s your proof that the insertion changed the plasmid. Different pattern, confirmed

“Okay, then it says the purpose of each plate in the pGLO experiment. So negative pGLO LB means what? It has no pGLO and no LB? What the fuck is LB again? And if it's negative, it means what? It has neither, or like, what does that mean?”

00:15

So “LB” is just the nutrient

Transcript Unavailable

00:00

Of course! “Negative pGLO LB” is your control plate—just normal bacteria on their nutrient “food” (LB). Since it’s “negative pGLO,” there’s no plasmid—so it shows how normal bacteria grow without any transformation. It’s basically your baseline to compare everything else!

“Okay, then what's your negative p-glow LB slash AMP or Amazon? Okay, so wait, your negative p-glow LB is no bacteria. That's your control group. Your negative p-glow LB and AMP is no p-glow, but you have AMP, but it's negative on both. I'm confused. Why is this saying negative on both and then positive on both? Because wouldn't that mean that your negative p-glow LB AMP is your control and so is your positive p-glow LB slash AMP? You're gonna have those two controls. And then the first one, which is negative p-glow LB, is gonna be like, it has nothing on it. So you can see what happens if there's like literally no bacteria. And then positive p-glow LB AMP and arabinose is gonna show those colonies in the difference.”

00:42

I hear you! Let’s break it down. “Negative pGLO” means the bacteria didn’t get the plasmid. “Positive pGLO” means they did. Now, LB is just food. If you have LB plus ampicillin (LB/amp) and no plasmid, those bacteria

Explain the purpose of each of the following plates in the pGLO experiment: –pGLO LB –pGLO LB/amp +pGLO LB/amp +pGLO LB/amp/ara For each plate, describe what growth (or lack of growth) indicates about transformation success and antibiotic selection.

The first plate, with no plasmid and just nutrient medium, shows normal bacterial growth. The second plate, with no plasmid but with ampicillin, will have no growth, showing the antibiotic works. The third plate, with the plasmid and ampicillin, shows that only transformed bacteria survive, so colonies mean transformation worked. The fourth plate, with plasmid, ampicillin, and arabinose, shows glowing colonies—proving transformation and that the GFP gene was switched on.

“Okay, can you explain to me the heat shock and transformation questions, where it's like explain why you use the CaCl2 transformation solution in your bacteria's suspension. Explain why heat shock is used during the transformation and explanation describe how temperature change affects the bacterial membrane. Then can you also explain to me what PBAD is?”

00:21

Sure! Calcium chloride helps make the bacteria’s membrane more permeable, making it easier for them to take up the plasmid. The heat shock is a sudden temperature increase—usually a quick warm-up—which briefly opens the membrane pores so the plasmid can slip inside. As for PBAD, it’s a promoter—a DNA region that acts like a start signal for the cell’s machinery to begin producing GFP when induced by arabinose.