Transformation AP 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!