pGLO and final thoughts on Neurosis article

Today you did the pGLO lab and wrote down your predictions.

Think about the following questions;

1. What genes are on the pGLO plasmid and what does each of them do?
2. What needs to be added to the E.Coli bacteria to make it grow and glow on LB with amp/ara added?
3. What will happen to the bacteria you plated (-pGLO, +pGLO) on the LB/ARA plate?
4. What are your predictions for each of your 8 plates?
5. What are the gene(s), trait(s), protein(s) invovled in this lab?
6. How does this relate to our question of the term? Environmental? Genetic? Both?

Finally, we talked about the neuroses article. Can you answer the questions below?

1. Is neurosis only genetic? If not give some examples of what environmental causes there are

2. Can  you explain the genetic cause discussed in the article? What is the gene, protein, trait?

3. How could you test if this gene really was involved in neurosis?

On your test:

Gene expression in Euk and Bacteria

Operons

Bacteria

Experimental Design

Neurosis

A brainy gene

Neuron HW

Enzymes

pGLO

Here are a couple videos to watch about pGLO. As you watch and do the experiment tomorrow, think about how this relates to our question of  "Is Biology Destiny?"






Watch this video up to 2:52

A Brainy Gene, The Neurosis article, and control of gene expression in eukaryotes

Monday we talked about the Brainy Gene article

Gene: NDE1
Protein: NDE1
Trait: formation of the cerebral cortex by changing how many and what cells are made.

A deletion mutation (2 different versions) causes a frame shift in the sequence. What does this do to the protein?

These mutations result in a much lower growth of the cerebral cortex resulting in children with brains that are only 10% of the normal size. These mutations are recessive therefore how many copies of the allele does there need to be for the microcephaly to be expressed?

GENE EXPRESSION -Control in Eukaryotes
How can gene expression be controlled in Eukaryotes?
-Reguation of mRNA
-Introns/Exons splicing
-Methylation
-Transcription Factors


We will focus on Transcription Factors(TF) (this relates to your Neurosis Article)
What are TF's? - proteins that bind to specific sequences of DNA upstream of a gene (these are the sequences that can be short or long that your neurosis article talked about), that increase or decrease the transcription of a gene.
-TF change expression by:
    -Changing shape of DNA - to allow easier binding of the RNA polymerase resulting in increased expression
    - Blocking genes from RNA polymerase - this lowers (turns off) gene expression.

Neurosis Article
In this article you read about a gene called 5HTTP which is a transport protein that is responsible for uptake of the neurotransmitter seratonin. which is involved in anxiousness. Scientists have discovered a region above the gene itself that along with transcription factors determines how many transporters are made and how quickly they take up seratonin. This region has 2 variants, one short and one long. If you have the short version, there is less receptors transcribed and therefore less uptake of seratonin leading to increased anxiousness, while the long version allows more transporters to be made, lowering the amount of seratonin and decreases anxiousness.

Does this work mean the trait of 'anxiousness' is only genetic, only environmental or both? What is your evidence?

The Lac operon continued and other bacteria operons

We began discussing gene expression. Can you answer the questions below?

If every cell has the same DNA sequence, how do cells differ?

In Bacteria how is gene expression controlled?
ex. Lac operon - set of inducible genes involved in digestion of lactose. (can you explain the lac operon?)

Other Operons in bacteria

AraC Operon: Involved in the digestion of Arabinose.

When no arabinose present, the AraC repressor is bound to the operator and this prevents the RNA polymerase from binding therefore, no transcription of the "Arabinose Utilization Genes." When Arabinose is present, Arabinose binds to the AraC repressor which changes shape (but unlike the Lac Operon respressor), it stays bound, but moves out of the way which allows RNA Poly to come in and bind and transcribe the genes.

Trp Operon: Involved in the synthesis of Tryptophan (an amino acid)

When tryptophan is present, it is bound to the repressor (making the repressor "Active"). This keeps the repressor bound to the operon, thereby preventing transcription of the tryptophan synthesis genes. When there is no tryptophan in the environment, tryptophan is not bound to the repressor. The repressor changes shape and unbinds from the operon, allowing RNA polymerase to come in to transcribe the synthesis genes which will make tryptophan.


Can you answer the questions below about operons?
1. How are the 3 operons different?
2. How are they similar?
3. Why would bacteria not want to make proteins in certain conditions (ex. if there is no arabinose present in the environment?)
4. What does this have to do with our question of the term? (. how does the environment of the bacteria increase/decrease the transcription of certain genes which determines the 'trait'  ex. being able to digest arabinose).


Operon video

Bacteria notes continued

On Saturday we discussed the ways bacteria can grow. Remember they grow by binary fission!

What are the 3 ways bacteria can share genetic material? (Transduction, Transformation, and Conjugation)

Can you explain each of the 3 ways?

How do we know natural selection has acted on antibiotic resistance in bacteria?

1. Heredity - bacteria inherit the plasmid where the genes for resistance are found

2. Variability - resistance vs. non-resistance

3.Selective Pressure - Antibiotic

4. Differential Survival and Reproduction - In the presence of antibiotics (In the environment), if the bacteria has a gene for resistance it will survive and reproduce. 

Bacteria shapes:

How bacteria grow?
Example of a lawn

Example of colonies

The Lac Operon

The Lac Operon is from E.Coli bacteria and is an example of an inducible set of genes, that can be turned 'on' and 'off' as needed (for example if lactose is present in the environment!)

The genes of the Lac Operon are responsible for breaking down the sugar Lactose from the environment the bacteria may be in and converting it into usable sugars for cell respiration.

Expression of the genes of the lac operon is determined by the presence or absence of Lactose.

Molecules involved:

Operator - part of the promoter. short sequence to which the repressor binds to inhibit transcription by RNA Polymerase. This binding is what stops the RNA Polymerase from binding to the rest of the promoter.

Promoter - Where RNA Polymerase binds to begin transcription after removal of the repressor protein

Repressor - protein that is bound to the operator in the absence of the sugar lactose. When lactose is present, it binds to the repressor and removes it from the operator. This allows the RNA Polymerase to bind to the promoter to begin transcription

Lactose - sugar involved in this operon. Binds to the repressor to remove it from the operon, to allow for transcription of the 3 genes that are involved in allowing the bacteria to use lactose for energy. 

Lac Z, Y, A - 3 genes involved in the process of importing and breaking down of lactose for energy. One gene is involved makes the transport protein to increase the amount of lactose within the cell (from it's ENVIRONMENT!). The other genes are involved in the breakdown of lactose into smaller sugars. 

This video (to 5:00) is a good explanation of the lac operon

So now can you answer the questions below?

1. What are the molecules involved in the lac operon?

2. Describe the steps of gene expression of the lac operon?

3. How does this relate to our question of the term?

Bacteria - transfer of genetic material

Thursday we will continue discussing bacteria and how their genetics AND their environment affect their ability to grow and survive.

Bacteria have 3 ways to increase their genetic diversity (for example 3 ways to gain resistance to an antibiotic)

1. Conjugation - 2 bacterial cells temporarily join and directly transfer genetic material between them (Textbook)

2. Transduction - involves viruses that infect bacteria. Virus carries genes from one cell to another

3. Transformation - Bacteria take up pieces of DNA from the ENVIRONMENT.

What do bacteria need to grow?

Nutrients such as: amino acids and sugars, Time

Space, Oxygen (some bacteria), pH, temperature (favorable environments)

Monday, quiz and pineapple wrap up

Today you had your quiz and then we wrapped up the pineapple lab. Make sure you can answer the following questions about enzymes.

1. What are enzymes?
2.What is an active site?
3. What things in the enzyme's environment can denature it?
4. What is the molecule an enzyme acts on?
5. How does the environment affect an enzyme's function?
6. What happens to the collagen in gelatin when you dissolve it in hot water and then refridgerate it?


We then went over the basics of bacterial growth (which is by binary fission).Here is a great site on bacterial growth and the stages.

From the cellsalive site:

LAG PHASE: Growth is slow at first, while the "bugs" acclimate to the food and nutrients in their new habitat.

LOG PHASE: Once the metabolic machinery is running, they start multiplying exponentially, doubling in number every few minutes.

STATIONARY PHASE: As more and more bugs are competing for dwindling food and nutrients, booming growth stops and the number of bacteria stabilizes.

DEATH PHASE: Toxic waste products build up, food is depleted and the bugs begin to die.

Homework for Thursday: Read and take notes on the 13.5 handout and then read the lab for thursday.

Homework for Monday

Check VC for homework!

Design lab - Enzymes and how they work

On Tuesday you designed your own lab to test if different fruit juices had proteolytic enzymes. Your reasoning was that like pineapple juice, orange, grapefruit, and lemon were all acidic and that could correlate with the presence of the enzyme.

Below is your Protocol
Materials
Pineapple HT/RT, Orange HT/RT, Grapefruit HT/RT, Lemon (pasteurized) HT/RT.
9 Test tubes
Hotplate, beaker with water
Gelatin (source of the collagen)
Kettle to heat water
Thermometer
Clamp
Water
Tape, Marker

Methods
1. Label test tubes (Pineapple HT/RT, Orange HT/RT, Grapefruit HT/RT, Lemon (pasteurized) HT/RT, water)
2.  Prepare Gelatin, mix in 1 packet of gelatin with 1/4 cold water and stir to somewhat dissolved. Then add in 3/4 cup of hot/boiling water and stir till all dissolved. Let cool to RT.
3. Turn on hot plate to high
3. Pipette 3 mL of each juice into appropriate tubes
4. Place the tubes labeled HT into the beaker on the hot plate. When the temp reaches 85C remove the tubes and cool them to RT
5. Add 10 mL of gelatin to each tube (both RT and HT)
6. Place tubes in rack then in fridge
7. Clean up your bench space!


Results!
Below are your results. Can you answer the following questions?

Pineapple design results from lvilleDrFox


1. What claim can you make based on the results and the results from your previous lab?
2. What is your evidence for this claim?
3. How valid is your research? How can you make it better?
4. If you wanted to redesign the experiment, what would you change?