You are introducing a foreign gene — the gene for Green Fluorescent Protein (GFP), originally from a jellyfish — into E. coli bacteria using a plasmid called pGLO. This process is called genetic transformation. You then use antibiotic selection and arabinose-induced gene expression to prove the transformation worked. The glowing green bacteria at the end are living proof that DNA from a jellyfish is now being read and expressed by a bacterium.
This experiment demonstrates the central framework of all molecular biology: > DNA → RNA → Protein → Trait > (pGLO gene → mRNA → GFP protein → green fluorescence)
The pGLO plasmid carries THREE key elements:
| Element | Gene/Feature | Function |
|---|---|---|
| Antibiotic resistance gene | bla (beta-lactamase) | Breaks down ampicillin → transformed cells survive on LB/amp plates |
| Fluorescent protein gene | GFP | Makes cells glow green under UV — but only when arabinose is present |
| Gene regulation system | araC + PBAD promoter | Controls WHEN GFP is expressed — the on/off switch |
| Origin of replication | ori | Allows the plasmid to replicate inside the bacterial cell |
Step 1 — Incubation (on ice) - Cells are mixed with CaCl₂ and pGLO plasmid - Ice temperature (0°C) slows everything down — keeps cells stable and allows slow, stable association of DNA with membrane - The cold + CaCl₂ together prime the cells for DNA uptake
Step 2 — Heat Shock (42°C, exactly 50 seconds) - The sudden jump from 0°C → 42°C creates a thermal imbalance - The rapid temperature change destabilizes the cell membrane, creating temporary pores or channels - The plasmid DNA is driven through these temporary openings into the cell - The transfer back to ice IMMEDIATELY after closes the membrane again - Timing is critical — too long at 42°C kills the cells
Step 3 — Recovery (room temp, LB broth, 10 min) - LB nutrient broth added provides nutrients for the stressed cells to recover - Cells begin repairing their membranes - Successfully transformed cells begin EXPRESSING their newly acquired genes — producing beta-lactamase (ampicillin resistance) so that when plated on ampicillin, they can survive
Constitutive expression = a gene that is ALWAYS being expressed, regardless of environmental conditions. No switch — it’s permanently “on.” - The bla gene (beta-lactamase/ampicillin resistance) on pGLO is constitutively expressed - As soon as the bacteria take up the plasmid, they start making beta-lactamase constantly - This is why transformed cells can immediately grow on ampicillin plates
Inducible expression = a gene that is only expressed when a specific signal (inducer) is present. It has a switch — it’s “off” by default and only turns “on” when triggered. - The GFP gene on pGLO is inducibly expressed - The inducer is arabinose (a sugar) - Without arabinose → GFP gene is OFF → no green glow → white colonies - With arabinose → GFP gene is ON → GFP produced → brilliant green colonies under UV
This is the molecular switch controlling GFP expression. Here are the key players:
| Player | What it is | What it does |
|---|---|---|
| PBAD | Promoter | The “landing pad” for RNA polymerase; sits upstream of GFP gene |
| araC | Regulatory protein | A DNA-binding protein that controls whether RNA polymerase can bind PBAD |
| Arabinose | Sugar (inducer) | The signal molecule that triggers the switch |
| RNA polymerase | Enzyme | Transcribes the GFP gene into mRNA |
Without arabinose: araC protein binds to the PBAD promoter in a shape that BLOCKS RNA polymerase from binding. GFP gene is NOT transcribed. No GFP protein made. Colonies appear white.
With arabinose: Arabinose enters the cell and binds directly to araC protein. This causes araC to CHANGE SHAPE. The new shape of araC actually HELPS RNA polymerase bind to PBAD. RNA polymerase transcribes the GFP gene into mRNA. mRNA is translated into GFP protein. Cells glow brilliant green under UV light.
Memory trick: Arabinose = key that unlocks araC = opens the door for RNA polymerase = GFP gets made = GLOW
This is the section that confuses everyone. Master this table and you master the experiment.
| Plate | Contents | Has pGLO? | Has ampicillin? | Has arabinose? | Expected growth? | Fluorescence? | Why? |
|---|---|---|---|---|---|---|---|
| +pGLO LB/amp | Transformed cells | YES | YES | NO | YES — colonies | NO (white) | bla gene gives amp resistance; no arabinose so GFP off |
| +pGLO LB/amp/ara | Transformed cells | YES | YES | YES | YES — colonies | YES (green glow) | bla gene gives amp resistance AND arabinose switches GFP on |
| -pGLO LB/amp | Non-transformed cells | NO | YES | NO | NO growth | NO | No plasmid = no bla gene = ampicillin kills all cells |
| -pGLO LB | Non-transformed cells | NO | NO | NO | YES — heavy growth | NO | No antibiotic = nothing killing cells = everything grows |
+pGLO LB/amp → grows, white colonies These cells successfully took up the pGLO plasmid. The plasmid carries the bla (beta-lactamase) gene which is constitutively expressed — meaning the bacteria immediately start producing the enzyme that destroys ampicillin. So even though the plate has ampicillin, these transformed cells can neutralize it and survive. No arabinose on this plate, so GFP is never switched on → white colonies, not green.
+pGLO LB/amp/ara → grows, GLOWING GREEN colonies Same transformed cells as above — they survive ampicillin because of the bla gene. But NOW arabinose is also present in the agar. The arabinose enters the cells, binds to araC protein, araC changes shape, RNA polymerase binds to PBAD, GFP gene is transcribed and translated → GFP protein accumulates → cells glow brilliant green under UV. This is the most exciting plate — proof that a jellyfish gene is being expressed in bacteria.
-pGLO LB/amp → NO GROWTH (negative control) These cells never received the plasmid. Without pGLO, they have no bla gene and cannot produce beta-lactamase. Ampicillin destroys the bacterial cell wall → cells die. No survivors, no colonies. This plate is the NEGATIVE CONTROL — it confirms that ampicillin is working and that any growth on the +pGLO/amp plates is truly due to transformation, not contamination or natural resistance.
-pGLO LB → grows, heavy growth (positive viability control) These cells never received the plasmid, and there is no antibiotic in the agar. Nothing is killing the cells. They grow freely and produce a lawn of bacteria — too many to count (TNTC). This plate serves as the POSITIVE CONTROL for cell viability — it confirms that the bacteria were alive and healthy going into the experiment. If this plate showed no growth, it would mean the bacteria were dead before transformation even started, and all other results would be meaningless.
Compare -pGLO LB/amp (no growth) vs. +pGLO LB/amp (growth). The ONLY difference between these two plates is whether pGLO was added. Both have ampicillin. Both use the same bacteria. The only explanation for growth on the +pGLO plate is that the plasmid was successfully taken up and the bla gene is being expressed. That IS transformation.
Transformation efficiency tells you how many bacterial cells were successfully transformed per microgram of DNA used. It is expressed as transformants per µg of DNA.
Transformation efficiency = colonies on LB/amp plate
────────────────────────────────────
µg of pGLO DNA spread on that plateStep 1: Total amount of pGLO DNA used
Total DNA (µg) = concentration (µg/µl) × volume of DNA added (µl)In the real experiment: 0.05 µg/µl × 5 µl = 0.25 µg total
Step 2: Fraction of DNA actually spread on the plate
Fraction = volume spread on plate (µl) ÷ total volume in tube (µl)Total volume in tube = 200 µl cells (from -pGLO) + 200 µl cells (split) + 5 µl DNA + 200 µl LB broth = 405 µl total Fraction = 100 µl ÷ 405 µl = 0.247
Step 3: µg of DNA spread on the plate
DNA spread = total DNA (µg) × fraction= 0.25 µg × 0.247 = 0.0617 µg
Step 4: Transformation efficiency
Efficiency = colonies on LB/amp ÷ µg DNA spreadUsing class data of 50 colonies: 50 ÷ 0.0617 = ~810 transformants/µg
Given: concentration = 4×10⁻³ µg/µl, volume DNA = 20 µl, LB broth = 200 µl, cells = 560 µl split into two (280 µl each), spread volume = 50 µl, colonies = 240
Q1. HB101 is naturally antibiotic resistant — True or False? ✅ FALSE HB101 is NOT naturally antibiotic resistant. This is the whole point of the experiment — ampicillin resistance only comes from taking up the pGLO plasmid (which carries the bla gene). If HB101 were already resistant, growing on ampicillin plates would prove nothing about transformation.
Q2. Which of the following is a method for making cells competent? ✅ All of the above — Protoplast formation, electroporation, AND microprojectile bombardment are all valid methods. In this experiment we use CaCl₂ + heat shock, but all four methods work by making the cell membrane permeable to DNA.
Q3. Matching — functions in our transformation experiment: - Makes cells permeable to plasmid DNA → A. CaCl₂ (neutralizes charge on membrane and DNA) - Selects for growth of transformed cells → B. Ampicillin (kills non-transformed cells, only transformed cells with bla gene survive) - Provides the GFP gene → C. pGLO plasmid (carries the engineered GFP gene) - Induces GFP gene expression → E. Arabinose (binds araC → changes shape → allows RNA polymerase to bind PBAD)
Q4. To increase transformation efficiency, cells are placed in 42°C water bath during which step? ✅ Heat shock The 42°C bath is the heat shock step. The rapid temperature change from 0°C to 42°C destabilizes the membrane, creating temporary channels through which the plasmid DNA enters. The shock must be exactly 50 seconds — too short and not enough DNA enters, too long and cells die.
Q5. In pGLO, PBAD controls expression of which gene? ✅ GFP PBAD is the promoter from the arabinose operon. In pGLO, the araB, araA, and araD genes have been REPLACED with the GFP gene. So PBAD now controls GFP instead of the arabinose-digesting enzymes. This is the genetic engineering that makes the experiment work.
Q6. Expression of the beta-lactamase gene (bla) from the plasmid is constitutive — True or False? ✅ TRUE The bla gene is constitutively expressed — it is always on, no inducer needed. As soon as the plasmid enters the cell, beta-lactamase is produced continuously. This is why transformed cells can immediately grow on ampicillin plates without needing any additional signal.
Genetic transformation is the process by which a cell takes up exogenous (foreign) DNA from its environment and stably incorporates it, potentially resulting in a change in genotype and phenotype. In bacteria, this natural process is exploited in the lab by making cells competent (permeable) and providing them with specific plasmid DNA. The transformed cells then express the genes carried on the foreign DNA as if those genes were their own.
In this experiment: E. coli HB101 (normally sensitive to ampicillin and non-fluorescent) takes up pGLO plasmid and becomes ampicillin-resistant AND capable of producing GFP (glowing green in the presence of arabinose).
Constitutive expression: A gene is expressed constantly regardless of environmental conditions. The cell always makes the protein. There is no regulatory switch — transcription proceeds at a steady rate all the time. - Example in this experiment: bla gene (beta-lactamase). Transformed bacteria produce beta-lactamase continuously from the moment the plasmid enters the cell. This is why they grow on ampicillin plates right away without needing any added sugar or signal.
Inducible expression: A gene is only expressed when a specific inducer molecule is present. Without the inducer, transcription is blocked. With the inducer, transcription is activated. - Example in this experiment: GFP gene. The GFP gene is controlled by the araC/PBAD system. Without arabinose, araC blocks transcription → no GFP → white colonies. With arabinose, araC changes shape and activates transcription → GFP produced → green fluorescence.
Why does this matter? Cells conserve energy by only making proteins when needed. It would be wasteful to produce GFP all the time — so the arabinose system acts as a smart switch, only turning GFP production on when arabinose (the inducer) is actually present.
E. coli HB101 (the host strain): - Non-pathogenic K-12 strain — safe for lab use - NOT naturally ampicillin resistant (makes selection meaningful) - NOT naturally competent (must be made competent with CaCl₂) - Does NOT naturally produce GFP - Grows well at 37°C on LB medium - Cells from exponential growth phase give best transformation efficiency
pGLO plasmid: - Small, circular DNA molecule (~5.4 kb) - Contains: GFP gene (from jellyfish Aequorea victoria), bla gene (ampicillin resistance), araC gene (regulatory protein), PBAD promoter (controls GFP expression), ori (origin of replication — allows plasmid to replicate inside bacteria) - GFP expression is inducible by arabinose (via araC/PBAD system) - bla expression is constitutive (always on) - Engineered by replacing araB, araA, araD genes with GFP gene in the arabinose operon
a) Why do we use LB/amp media to select for transformants?
Ampicillin is an antibiotic that kills bacteria by blocking cell wall synthesis. E. coli HB101 is naturally sensitive to ampicillin — it will die on LB/amp plates. HOWEVER, the pGLO plasmid carries the bla gene which encodes beta-lactamase — an enzyme that breaks down and inactivates ampicillin. Transformed cells express beta-lactamase constitutively, neutralizing the ampicillin around them and surviving. Non-transformed cells have no bla gene, cannot neutralize ampicillin, and die.
Result: LB/amp plates are a SELECTIVE medium — only transformed cells can grow. This is called selection with a selectable marker (the bla gene is the selectable marker). It filters out the approximately 99.9% of cells that didn’t take up the plasmid, leaving only the transformed bacteria visible as colonies.
b) What was the purpose of arabinose?
Arabinose is the INDUCER of GFP gene expression. In pGLO, the GFP gene is controlled by the araC/PBAD system from the arabinose operon. When arabinose is present: 1. It enters the bacterial cell 2. It binds to the araC regulatory protein 3. Arabinose causes araC to change its 3D shape (conformation) 4. The new araC shape promotes (helps) RNA polymerase binding to the PBAD promoter 5. RNA polymerase transcribes the GFP gene into mRNA 6. mRNA is translated into GFP protein 7. GFP protein accumulates → cell fluoresces brilliant green under UV light
Without arabinose, araC stays in its default shape which blocks RNA polymerase from binding PBAD → GFP gene is never transcribed → no fluorescence.
c) Which plate — LB/amp or LB/amp/arabinose — produced glowing colonies? WHY?
LB/amp/arabinose produced the glowing green colonies.
The LB/amp plate also has transformed cells (with pGLO), but those cells appear WHITE — not glowing. This is because there is no arabinose present, so the GFP gene is never switched on even though the cells have it.
The LB/amp/ara plate has the same transformed cells PLUS arabinose in the agar. The arabinose enters the cells, triggers the araC conformational change, activates PBAD, and GFP is produced in large quantities. The cells fill up with GFP protein and glow brilliant green under UV.
Key insight: BOTH the LB/amp and LB/amp/ara plates have transformed cells. The difference is ONLY whether arabinose is present. This elegantly demonstrates that the cells have the GFP gene (they took up pGLO) but only express it when the inducer is present.
d) Which plates are the control plates? What are their purposes?
Negative control: -pGLO LB/amp Contains non-transformed cells on ampicillin. Should show NO growth. Purpose: confirms that (1) ampicillin is working properly and (2) HB101 cells are NOT naturally ampicillin resistant. Any growth here would indicate contamination or a problem with the ampicillin. This is what lets you claim that growth on the +pGLO/amp plate is due to TRANSFORMATION, not some other factor.
Positive viability control: -pGLO LB Contains non-transformed cells on plain LB (no antibiotic). Should show HEAVY growth (lawn/TNTC). Purpose: confirms that the bacteria were alive and healthy at the time of plating. If this plate showed no growth, it would mean your bacteria were dead before transformation started, and ALL your results would be invalid — including the +pGLO plates. You need to know the bacteria were viable.
See Part 4 above for the complete step-by-step formula and worked examples.
Key concepts: - Transformation efficiency is expressed as transformants per microgram (µg) of DNA - You count colonies on the LB/amp plate (not LB/amp/ara, because not all transformed cells may express GFP depending on arabinose concentration) - You calculate how much DNA was actually spread on that specific plate (not the total DNA used in the whole experiment) - Higher efficiency = better transformation procedure (better competent cells, better heat shock, less DNA degradation)
| Plate | Expected | Data from 25/26 | Error? | Explanation |
|---|---|---|---|---|
| +pGLO LB/amp | Colonies (some) | 0 | YES | Transformation failed — either DNA not added, heat shock not done, or cells were dead |
| +pGLO LB/amp/ara | Colonies + glow | 50 (no fluorescence) | YES (partially) | Colonies grew (transformation worked) but NO fluorescence — arabinose likely missing from agar, or wrong plate used |
| -pGLO LB/amp | No growth | 0 | NO | Correct — no plasmid, no amp resistance, no growth |
| -pGLO LB | Heavy growth | TNTC | NO | Correct — no antibiotic, bacteria grow freely |
Error in +pGLO LB/amp (0 colonies): The LB/amp/ara plate shows 50 colonies, so transformation clearly worked. The most likely error for 0 colonies on LB/amp is that the wrong suspension was plated (e.g., -pGLO was accidentally put on this plate), or the plate itself had a problem.
Error in +pGLO LB/amp/ara (50 colonies, no fluorescence): Colonies grew = transformation worked = plasmid was taken up = bla gene expressed. BUT no fluorescence means GFP was NOT expressed. Most likely cause: arabinose was missing from the agar (wrong plate used, or the ara plate was mislabeled), OR the UV lamp was not used to check for fluorescence.
Answer each question. Check answers and rationale in Part 9.
Q1. What is genetic transformation?
Q2. Why is CaCl₂ used in bacterial transformation?
Q3. What is the purpose of the heat shock step at 42°C?
Q4. The bla gene on pGLO is constitutively expressed. What does this mean?
Q5. Which plate should show glowing green colonies?
Q6. Why does the -pGLO LB/amp plate show no growth?
Q7. What role does araC play in GFP expression?
Q8. The +pGLO LB/amp plate grows white colonies. Why are they WHITE and not green?
Q9. What is the purpose of the -pGLO LB plate (plain LB, no antibiotic)?
Q10. Which comparison BEST proves that genetic transformation occurred?
Q11. What is transformation efficiency?
Q12. If a student forgot to add arabinose to the LB/amp/ara plate, what would they observe?
Q13. In the pGLO plasmid, the araB, araA, and araD genes have been replaced with:
Q14. Which of the following is NOT a method for making bacterial cells competent?
Q15. What would happen if you spread +pGLO cells on a plain LB plate (no ampicillin, no arabinose)?
| # | Answer | Rationale |
|---|---|---|
| 1 | B | Genetic transformation is the uptake of foreign DNA by a cell, which can change its genotype and phenotype. It doesn’t involve mutation by radiation (that’s mutagenesis) or translation (that’s protein synthesis). |
| 2 | C | Both DNA and the bacterial membrane are negatively charged, so they repel each other. Ca²⁺ ions from CaCl₂ bridge these negative charges, neutralizing the repulsion and allowing DNA to approach and associate with the membrane. |
| 3 | C | The sudden shift from 0°C to 42°C creates a heat-driven destabilization of the membrane, temporarily forming channels or pores through which the plasmid DNA can enter. The rapid return to ice then closes these openings. |
| 4 | B | Constitutive expression means the gene is always “on” with no need for an inducer. The bla gene is expressed immediately and continuously once the plasmid is inside the cell, which is why cells can grow on ampicillin right away after transformation. |
| 5 | D | +pGLO LB/amp/ara is the only plate with BOTH conditions required for green fluorescence: the pGLO plasmid (providing the GFP gene) AND arabinose (the inducer that switches GFP expression on). |
| 6 | B | HB101 has no natural ampicillin resistance. Without pGLO, there is no bla gene and no beta-lactamase enzyme. Ampicillin destroys the bacterial cell wall and the cells die. |
| 7 | B | araC is a regulatory protein that binds DNA at PBAD. Without arabinose, araC blocks RNA polymerase. When arabinose binds araC, it causes a conformational (shape) change that allows RNA polymerase to bind PBAD and transcribe the GFP gene. |
| 8 | C | These cells DO have the pGLO plasmid (proven by their survival on ampicillin via the bla gene). They also DO have the GFP gene. But without arabinose, araC stays in its blocking configuration — RNA polymerase cannot bind PBAD — so GFP mRNA is never made. No GFP = no green = white colonies. |
| 9 | C | The -pGLO LB plate is a viability control. It shows that the bacteria used in the experiment were alive and capable of growing. Without this control, if all plates showed no growth, you couldn’t tell if transformation failed or if the bacteria were simply dead from the start. |
| 10 | B | The comparison between -pGLO LB/amp (no growth) and +pGLO LB/amp (growth) is the critical proof. The ONLY variable between these two plates is whether pGLO was added. Both use the same bacteria, same ampicillin plate. Growth only in the +pGLO condition proves that the plasmid (and specifically its bla gene) is responsible. |
| 11 | B | Transformation efficiency = number of successful transformants per µg of DNA used. It is a measure of how efficient your transformation protocol was at getting DNA into cells. |
| 12 | B | If arabinose is missing, transformation still works (cells still take up pGLO and grow on ampicillin via bla gene). But without the inducer, araC stays in blocking mode, RNA polymerase cannot transcribe GFP, and no GFP protein is made → white colonies, no fluorescence. |
| 13 | C | In the original arabinose operon, PBAD controls the araB, araA, araD genes that digest arabinose. In pGLO, these three genes were replaced by the single GFP gene. So PBAD now controls GFP instead of arabinose-catabolism genes. |
| 14 | C | Arabinose is an inducer of gene expression — it turns on the GFP gene. It has nothing to do with making cells competent (permeable to DNA). The methods for making cells competent are CaCl₂+heat shock, electroporation, protoplast formation, and microprojectile bombardment. |
| 15 | B | On plain LB with no ampicillin, transformed cells survive just fine (no antibiotic to kill them). They grow into white colonies. Without arabinose, GFP is never induced, so colonies are white not green. Heavy growth of white colonies. |
No answer choices — write your answers from memory!
FRQ 1. Define genetic transformation in your own words:
FRQ 2. What are the THREE key elements carried on the pGLO plasmid and what does each one do?
FRQ 3. Explain the difference between constitutive and inducible gene expression. Give one example of each from this experiment:
Constitutive: _______________________________________________________________
Inducible: _______________________________________________________________
Example of constitutive in this experiment: _______________________
Example of inducible in this experiment: _______________________
FRQ 4. Fill in the expected results for each plate:
| Plate | Growth? (yes/no) | Fluorescence? (yes/no) | Why? |
|---|---|---|---|
| +pGLO LB/amp | |||
| +pGLO LB/amp/ara | |||
| -pGLO LB/amp | |||
| -pGLO LB |
FRQ 5. Explain step by step what happens molecularly when arabinose is added to transformed bacteria:
FRQ 6. Why does the -pGLO LB/amp plate serve as the negative control? What specifically does it control for?
FRQ 7. Why does the -pGLO LB plate serve as the positive viability control? What would it mean if this plate showed NO growth?
FRQ 8. The +pGLO LB/amp plate grows white colonies instead of green. A student says “these cells must not have taken up the plasmid.” Is the student right? Explain:
FRQ 9. Describe the three steps of bacterial transformation in this experiment and explain what is happening at the molecular/cellular level during each step:
Step 1 — Incubation: _______________________________________________________________
Step 2 — Heat shock: _______________________________________________________________
Step 3 — Recovery: _______________________________________________________________
FRQ 10. Calculate transformation efficiency using the following data. Show all work with units: - DNA concentration: 0.05 µg/µl - Volume of DNA added: 5 µl - Total volume in tube at time of plating: 405 µl - Volume spread on LB/amp plate: 100 µl - Colonies counted on LB/amp plate: 75
Total DNA used = _______________
Fraction of DNA spread = _______________
µg DNA spread on plate = _______________
Transformation efficiency = _______________ transformants/µg
FRQ 11. A group’s +pGLO LB/amp/ara plate shows 50 colonies but NO fluorescence. List two possible explanations:
FRQ 12. Why is it important that HB101 is NOT naturally ampicillin resistant? What would happen to your results if it were?
FRQ 13. If you spread 100 µl of the +pGLO transformation solution onto a plain LB plate (no ampicillin, no arabinose), what would you see? Explain why:
FRQ 14. In the pGLO plasmid, what promoter controls GFP expression? What two things are required simultaneously for GFP to be expressed?
Promoter: _______________________
Required: 1. _______________________ 2. _______________________
FRQ 15. Explain why cells from the exponential growth phase give better transformation efficiency than cells from the stationary phase:
FRQ 1. Genetic transformation is the process by which a bacterial cell takes up foreign DNA (such as a plasmid) from its environment, incorporates it, and expresses new genes encoded by that DNA, resulting in new traits or phenotypes in the transformed organism.
FRQ 2. 1. bla gene (beta-lactamase) → provides ampicillin resistance; the enzyme breaks down ampicillin so transformed cells survive on LB/amp plates (constitutively expressed) 2. GFP gene → encodes Green Fluorescent Protein; causes cells to fluoresce green under UV light when expressed (only when arabinose is present) 3. araC gene + PBAD promoter → the regulatory switch; araC protein controls whether RNA polymerase can transcribe GFP based on presence/absence of arabinose
FRQ 3. Constitutive: a gene that is expressed at all times regardless of environmental conditions — always “on,” no inducer needed. Inducible: a gene that is only expressed when a specific inducer signal is present — “off” by default, switched “on” by inducer. Constitutive example: bla gene (beta-lactamase/ampicillin resistance) — expressed continuously from the moment pGLO enters the cell. Inducible example: GFP gene — only expressed when arabinose is present to activate the araC/PBAD system.
FRQ 4.
| Plate | Growth? | Fluorescence? | Why? |
|---|---|---|---|
| +pGLO LB/amp | YES | NO (white) | bla gene gives amp resistance so cells survive; no arabinose so GFP off |
| +pGLO LB/amp/ara | YES | YES (green) | bla gives amp resistance AND arabinose switches GFP on |
| -pGLO LB/amp | NO | NO | No plasmid = no bla gene = ampicillin kills all cells |
| -pGLO LB | YES (heavy) | NO | No antibiotic to kill cells; no arabinose; bacteria grow freely |
FRQ 5. 1. Arabinose enters the bacterial cell from the surrounding agar medium 2. araC protein binds arabinose, causing a conformational (shape) change in the araC protein 3. RNA polymerase can now bind to the PBAD promoter (araC in its new shape facilitates this binding) 4. GFP gene is transcribed into mRNA, then translated into GFP protein 5. GFP protein accumulates in the cell → cell fluoresces brilliant green under UV light
FRQ 6. The -pGLO LB/amp plate is the negative control because it contains bacteria that never received the plasmid, plated on ampicillin. It should show NO growth. It controls for natural antibiotic resistance — if these cells grew, it would mean HB101 has some natural resistance to ampicillin, which would invalidate the selection system. No growth here confirms that ANY colonies on the +pGLO/amp plates are truly due to transformation, not pre-existing resistance.
FRQ 7. The -pGLO LB plate confirms that the bacteria were alive and healthy going into the experiment. Heavy growth (TNTC) on this plate proves the bacteria were viable. If this plate showed NO growth, it would mean the bacteria died before or during the experiment — their cells were already dead. In that case ALL other plates (including +pGLO plates) showing no growth would not be meaningful, because dead cells can’t transform regardless of technique.
FRQ 8. The student is WRONG. The white colonies on the +pGLO LB/amp plate PROVE that those cells DID take up the plasmid. Here’s why: the plate contains ampicillin, which kills any cell without the bla gene. If the cells are growing on ampicillin, they must have the bla gene — which only comes from the pGLO plasmid. So these cells definitely have pGLO. The reason they’re white (not green) is because there is no arabinose present on this plate, so the GFP gene is never switched on even though the cells have it.
FRQ 9. Step 1 — Incubation: Cells are mixed with CaCl₂ and pGLO plasmid on ice. Ca²⁺ ions neutralize the negative charges on the bacterial membrane and on the DNA, allowing the DNA to associate with the outer surface of the membrane. The cold temperature keeps cells stable and slows everything down for gradual, stable DNA-membrane association.
Step 2 — Heat shock: The tube is rapidly transferred from 0°C ice to a 42°C water bath for exactly 50 seconds. This sudden temperature change destabilizes the phospholipid bilayer of the bacterial membrane, temporarily creating openings or pores. The thermal imbalance drives the plasmid DNA through these pores into the cytoplasm. The tube is immediately returned to ice to close the membrane and prevent cell death.
Step 3 — Recovery: LB nutrient broth is added and cells incubate at room temperature for 10 minutes. The nutrients allow stressed cells to repair their membranes. Transformed cells begin expressing their newly acquired genes — most importantly, they start producing beta-lactamase from the bla gene so that when plated on ampicillin, they can immediately neutralize it and survive.
FRQ 10. - Total DNA = 0.05 µg/µl × 5 µl = 0.25 µg - Fraction = 100 µl ÷ 405 µl = 0.247 - µg spread = 0.25 × 0.247 = 0.0617 µg - Efficiency = 75 ÷ 0.0617 = ~1,216 transformants/µg (≈ 1.2 × 10³ transformants/µg)
FRQ 11. 1. The agar plate was prepared without arabinose (wrong plate used, or arabinose was accidentally left out) — cells have pGLO and grew on ampicillin via bla gene, but without arabinose the GFP switch was never activated 2. The UV lamp was not used when checking for fluorescence, or the UV lamp was broken — fluorescence may actually be present but was not detected
FRQ 12. If HB101 were naturally ampicillin resistant, growing on ampicillin plates would tell you nothing about transformation. Both transformed (+pGLO) and non-transformed (-pGLO) cells would grow on LB/amp plates. You would lose the ability to select for transformed cells. The entire selection system depends on HB101 starting with NO resistance — so that resistance (from the bla gene) can ONLY come from taking up the plasmid.
FRQ 13. You would see heavy growth of WHITE colonies — a lawn similar to the -pGLO LB control plate. On plain LB with no ampicillin, all bacteria (transformed and non-transformed alike) can grow freely. There is nothing killing any of them. Without arabinose, GFP is never induced, so the transformed cells among them would still appear white (not green). You could not distinguish transformed from non-transformed cells on this plate.
FRQ 14. Promoter: PBAD Required: 1. araC protein (bound to DNA at PBAD) in the presence of arabinose (its conformational change allows RNA polymerase to bind) 2. Arabinose (the inducer that changes araC shape and activates the system)
FRQ 15. Cells in exponential (log) phase are actively dividing — their membranes are more fluid, dynamic, and metabolically active compared to stationary phase cells. This makes them more susceptible to the CaCl₂ and heat shock treatment and better able to take up and incorporate foreign DNA. Stationary phase cells have more rigid membranes and are metabolically slower, making transformation much less efficient. Also, exponentially growing cells are better equipped to immediately express newly acquired genes during the recovery step.