Anticipation of GfP pop quiz

Why is the heat shock step vital to the success of your experiment?

The heat shock step facilitates the entry of the plasmid DNA into the bacterial cells. If the plasmid does not get into the cells, you won’t see the new traits. It also is vital because it increases the permeability of bacterial cell membranes, facilitating the uptake of the plasmid DNA during transformation. This enhances the chances of successful integration of the desired genes into the bacterial genome, crucial for expressing the desired traits, such as GFP fluorescence in this experiment

Outline the steps that could be followed to use recombinant DNA technology to produce insulin.

Isolate the human insulin gene from human DNA using restriction enzymes and PCR.

2)Insert the insulin gene into a vector, such as a plasmid, using restriction enzymes and ligase.

3)Transform the plasmid containing the insulin gene into a host cell, like E. coli, using techniques like electroporation.

4)Allow the transformed host cell to replicate the plasmid and produce insulin.

5)Harvest the synthesized insulin from the host cell using purification techniques, such as column chromatography. 6)Assess the purity and activity of the insulin using techniques like SDS-PAGE and ELISA to ensure its safety and efficacy for human use

Brainstorm how you could use recombinant DNA technology to make normal E. coli bacterial cells glow green

To make E. coli cells glow green, we insert the GFP gene, which encodes the green fluorescent protein, into either the bacterial DNA or a plasmid. This involves cutting the DNA with restriction enzymes and ligating the GFP gene into place. This method, widely used to track bacteria in environmental systems, enables us to visualize their presence via green fluorescence.

Where in nature could you find a source for a glowing protein? Discuss your ideas with a partner

One example of bioluminescent proteins found in nature is the Vibrio fischeri, which emit light because of chemical reactions within their cells. In addition, Fireflies are another well-known source of exemplification of this, due to their luciferase enzymes producing light as part of their mating rituals.

summarize the goal of the experiment in a well-crafted paragraph in your laboratory notebook. Your summary should include the terms listed below. Underline each term

The objective of this experiment is to introduce the green fluorescent protein (GFP), sourced from the jellyfish, into E. coli bacteria using plasmid vectors. The GFP will be incorporated into the bacterial genome via chemical transformation, utilizing a method involving heat shock and calcium chloride solution. Plasmids containing the GFP gene will confer resistance to ampicillin, aiding in the selection of transformed bacteria. Upon successful transformation, the E. coli colonies expressing GFP will fluoresce green when exposed to ultraviolet light. The addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) will induce GFP expression, allowing for visualization of transformed colonies. Through this experiment, we aim to produce a population of E. coli bacteria expressing GFP, demonstrating successful genetic modification and the potential for various applications in biological research.

GFP Sequence:

This sequence encodes the green fluorescent protein (GFP), which fluoresces green when exposed to certain wavelengths of light, facilitating visualization of transformed cells

AmpR Sequence:

This sequence encodes resistance to the antibiotic ampicillin, allowing for the selection of bacterial cells containing the plasmid by enabling their survival in the presence of ampicillin.

T7 Promoter:

This sequence serves as the promoter region for the T7 RNA polymerase, facilitating transcription of the GFP gene from the plasmid into messenger RNA (mRNA), ultimately leading to the production of GFP protein within the bacterial cells.

Why will a plate with only -DNA not glow and not have any growth?

Without the presence of the plasmid carrying the GFP gene, there should be no growth or green fluorescence observed on this plate.

Why will there be growth but no glow for something with -DNA/+AMP have growth but not glow?

The presence of ampicillin will select for bacterial cells that have taken up the plasmid with the ampicillin resistant gene. However, without the GFP gene, there should be no green fluorescence observed

Why will something with +DNA/+AMP gave growth but no glow?

The presence of both DNA and ampicillin selects for bacterial cells containing the plasmid with the GFP gene. However, without the introduction of GFP expression (via IPTG), there should be no green fluorescence observed.

Why will the plate +DNA/+Amp/+IPTG have growth and a glow?

The presence of DNA, ampicillin, and IPTF selects for bacterial cells containing the plasmid with the GFP gene. IPTG induces the expression of GFP, leading to green fluorescence observed under UV light. Therefore, this plate should show both growth and green fluorescence