TransformationAP Big Idea #3: Genes and Information Transfer connected with AP Big Idea #1 (Evolution) & #2 (Cellular Processes)

How has our understanding changed?

What distinguishes stem cells from differentiated cells?

What types of genes are turned on/off as the path towards differentiation occurs?

Its all about regulation! Gene Regulation in Bacteria

Operons Use of bacterial gene regulation systems in

biotechnology

Gene Regulation in Eukaryotes: (We’ll come back to this in a later powerpoint!!!) Regulation at the DNA Level Regulation at the transcription level Regulation at the translation level

Bacterial Operons Operon: set of genes that control the

production of a certain protein product needed by the cell under specific conditions. Inducible Repressible

What do you think is the difference between these?

Inducible: Example LAC

How does the presence of lactose “induce” transcription?

Repressible: TRP operon

Why are operons necessary? What is the advantage of E. coli having

evolved? A lac operon? A trp operon?

Using Bacterial Genetics for Biotechnology Transformation:

Bacteria can uptake eukaryotic DNA and become gene factories.

What is a plasmid?

Basic Structure of a Plasmid

What does a plasmid do in a bacteria? “Sex”

Selectable markers Resistance to antibiotics (like kanomycin) If bacteria gets plasmid; it will grow in

presence of antibiotic. Screens out bacteria that don’t receive

plasmid.

Origin of Replication Bacteria divide

rapidly to form colonies.

They must be able to replicate the plasmid into their daughter cells.

Making a plasmid:

Restriction enzymes: Enzyme present in bacteria that cuts DNA

at certain points (used to protect bacteria from viruses)

Used in biotechnology to cut open a plasmid and glue in a gene of interest

Ligase: Enzyme used to glue in inserted piece of

DNA (where did we talk about this enzyme before?)

Problems that can be encountered

The hard part is done! We bought pre-engineered pGLO

plasmid. Plasmid was engineered and then

copied by BioRAD!

Engineering a bacteria

Steps we will do: Heat shock bacteria to add plasmid. Grow (clone) bacteria to give them a

chance to replicate plasmid (E. coli populations double every 20 minutes)

Incubate bacteria and allow them to produce protein=green fluorescence protein

Use an operon to turn on/off the presence of protein production!

pGLO Lab: How can we alter the transformation

process to improve transformation efficiency? Each lab group

What you must know before the lab: Research the pGLO plasmid. Be able to identify:

The operon, the selectable marker, the inserted gene. Read through the manual (available on BioRad’s

website and through a quick web search…pGLO student manual)

Predict what will happen on each plate you make: LB -pGLO LB/Amp -pGLO LB/Amp +pGLO LB/Amp/Ara +pGLO

Lab Day: Decide how will your group “tweak” the

procedure to attempt to increase transformation efficiency.

Identify the expected result for each plate and understand why each control is used.

Identify in your lab notebook how you will calculate transformation efficiency (for your plate that produces GFP) the next day.

Data and Analysis Day: Calculate control transformation

efficiency. Put in spreadsheet (we will have 3 sets of data for class control)

Calculate the average transformation efficiency for your 3 experimental titles.

In your groups, make sure you understand the specific purpose of your group’s lab!

Follow-up: This lab is a model of using transformation

to produce a protein product. Find a real life example of how bacterial

transformation was used (or how GFP was used) and explain understanding this model is useful (implications and connections!)

Remember…we are using Semester 2 Lab rubric…Here is a chance to show the skills you learned as a big lab group!