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?

Monday -Design lab

Today we reviewed your pineapple enzyme design labs and chose 2 possible ideas.

1. Independent Variable - juice type (orange, peach, grape, lemon).


2. Independent Variable - temperature (85, 60, 37, 24)

Part of your homework again tonight is to write up the PROCEDURE. You must have it written out when you arrive tomorrow.

Remember to use your original lab as a guide. You need to have a list of all the materials you need and then the steps (i.e. how to make the jello, how much to add to each tube, how many tubes does each group need). AND then a list of the IV, DEP, etc.

example from your original lab

1.      Label test tubes as “pineapple – hot,” “pineapple – RT,” “apple juice – hot,” “apple juice – RT,” and “water.”  Apply the tape at the top of the test tube with a “tail” as shown at right, so steam from the water bath does not remove the labels.

2.      Prepare gelatin in an appropriate beaker.

a.    Dissolve the contents of a gelatin packet in ¼ cup cold water.

b.    Add ¾ cup boiling water to the mixture and stir until completely dissolved.

3.      Pipette 3ml of juice or water into each test tube according to the labels, using transfer pipettes.  Do not cross-contaminate juices by using a pipette in different types of juice.

4.      Place your “hot” tubes in a beaker of water.  Heat the beaker on the hotplate until a thermometer in the water of the beaker reads 85°C.  Carefully remove the tubes from the heat.  Allow contents to come to room temperature (RT).

5.      Add 10 ml ROOM TEMPERATURE gelatin mixture to each test tube.  Use the big pipette for this and do not let the gelatin drip into the green pipetter.  Shake tubes well to ensure mixing of contents

6.      Refrigerate test tubes overnight.

We also reviewed the sickle cell gel electrophoresis handout. If asked could you answer the following questions..
1. What is the phenotype if someone is Aa? Explain
2. What is a recessive allele?
3. How do we use restriction enzymes to determine genotype of sickle cell?

The last part of your homework is the handout below

7 text homework 5 from lvilleDrFox

Thursday - More on Enzymes

Thursday we worked on the lab involving how proteolytic enzymes and/or heat affect proteins such as collagen. Collagen is a structural protein involved in skin, connective tissue and bones of animals. Below are a couple of images of collagen and how it is organized.

Enzymes have specific 'substrates' or targets on which they work. Some enzymes 'digest' or 'break down' their substrate, while others put 2 targets together. Which type is our 'proteolytic' enzyme from fruit? What is the enzyme's substrate in our lab? Will that enzyme break down another substrate?

Proteins (which is what enzymes are!) can be denatured by heat and some chemicals. This is the process of denaturation.

In our lab, we are looking how 2 variables affect

More on Restriction Enzymes and Gel Electrophoresis

Today we reviewed Restriction Enzymes and Gel Electrophoresis.

Homework for tomorrow: handout from today and prelab questions

Here is a great site for more practice with gel prediction, plasmids, and restriction enzymes.
Practice Reading a Gel (you can do the entire game for more instruction).

Here is a video that also takes you through a tutorial of what we did today.

Homework for Monday

Please read the article below and watch the video(s) and answer the questions provided. Remember..."is biology destiny?"

15 a life without fear from lvilleDrFox

Here is the video

Friendly to the Extreme: Meet Kids and Adults With Williams Syndrome - ABC News

Mutations/Protein Folding and Homework for tomorrow.

Today we reviewed translation and then talked about the videos you watched on Mutations and Protein Folding

HOMEWORK for tomorrow: Finish your electrophoresis lab

Mutations:

A change in the DNA sequence (Nucleotides or larger changes).

Can you identify the Fact or Fiction and explain?

¨ All mutations are bad…………………….¨ Mutations are a source of evolution…¨ Mutation can be silent……………………¨ Mutations only happen when DNA is exposed to things like radiation………………………..


What are the major types of mutation?

Point Mutations
Substitution - single nucleotide change 
Insertion - a base pair is added in the sequence
Deletion - a base pair is removed from the sequence

Both insertion and deletion can also be large chunks put in or removed

Duplication - a large section of DNA on a chromosome is copied. Is this always bad? Can the new portion gain a new function (after a LARGE time frame)

Translocation - the transfer of a piece of one chromosome to a nonhomologous chromosome. Remember the two chromosomes involved typically exchange pieces.

Can you answer the questions below?
1. What is the difference between Substitution and Insertion? Duplication and Translocation?
2. What is a silent mutation?
3. How is mutation involved in evolution?
4. What disease did we discuss that is a change in only 1 nucleotide and is the mutation only bad or does it help?
5. When will a mutation be heritable?

Protein Folding:

Today we talked about how the order and type of the amino acid in a polypeptide determines the folding structure of the protein

What are the 4 levels of protein folding?

Primary - string of amino acids

Secondary - alpha helix or beta pleated sheet

Tertiary - final protein folding (+/- aa come together, hydrophobic aa go on the inside, hydrophillic go on the inside)

Quaternary - multiple proteins fold/come together to form complexes

Here is a good website to try more protein folding

Types of amino acids:
1. Charged (+/-)
2. Polar vs. Nonpolar
3. Special Cases

Restriction Enzymes

Today we discussed Restriction Enzymes and Gel Electrophoresis.

Watch the video from start to 9:28 and then using the notes from today and the video:
 Can you answer the following questions?
1. In what organism do you find restriction enzymes?
2. What are restriction enzymes used for in that organism?
3. What are the nucleotide sequences that a restriction enzyme looks for called?
4. Why does the organism's own DNA not get cut into pieces?
5. What are the small circular pieces of DNA called in the organism and what is usually found on these circles?


We also went over how to tell band size resulting from plasmids being cut by restriction enzymes.

Here is a video (watch till 4:50) on how to read a gel


 We also began talking about Gel Electrophoresis. (remember this is also in the above video)

Here is the virtual lab link
Can you answer the questions below?
1. What does GE do?
2. What type of charge does DNA have?
3. What do the black and red cords represent?
4. Which DNA 'runs' closer to the bottom (small or large)?

Here is the picture you will use from your lab to answer the postlab questions (the wells are on the left)

Timid Mice

Today we discussed the timid mice article. You will get more practice reading portions of a scientific article. I've posted a powerpoint. Can you fill out the table on the second slide?

The article discusses how stathmin affected the fear response of mice.

Translation and Mutation ppt

Here is the translation and mutation part of your powerpoint slides from class. Please do read through/print them out.

Translation mutation ppt from lvilleDrFox

Thursday Translation continued

On Thursday you had a 5 minute quiz on the differences and similarities between transcription and translation. (make sure you can explain these)

Remember DNA (a gene)--->mRNA--->Protein--->Trait. Where does environment come into play?


You then drew the process of translation up on the board. Here are the steps of translation: Make sure you can explain the whole process!

1. mRNA exits the nucleus
2. mRNA goes to the ribosome (in the cytoplasm) and ribosome attaches to the mRNA
3. mRNA moves through the ribosome 1 codon at a time
4. tRNA comes in and reads codon, if the anticodon is the correct one (with the complementary bases) the amino acid gets added to the peptide chain (remember the first codon read will always be the start codon... what amino acid does this code for?)
5. The amino acids are covalently bound to each other (this is called a peptide bond)
6. Once the ribosome gets to the stop codon of the mRNA, this is the signal to stop translation.
7. The protein (peptide chain) is released from the ribosomes and tRNA
8. The protein is then folded in a 3 dimensional structure.

Think about the following questions:
Describe the process of translation
How does translation relate to making proteins?
Where does translation take place?
What is the result of translation?
What do proteins do?
How does this relate to "Is Biology Destiny?"

Also here is the correct video for the HHMI video of translation


Can you transcribe then translate the following sequence?
TAC CCG GTA TTA CAG TGG GAA TGA ATT

Tuesday - DNA extraction and Beginning Translation

Today we did the Strawberry DNA extraction. You should be able to answer the questions below!

1. What are the following used for?
             Strawberry (why can't we use another fruit?)
             Soap/Salt
             Ethanol

2. How is DNA organized in the Cell?

3. Why does the DNA tangle when we remove it from the nucleus?

Here is good video describing the experiment!



We also started discussing translation. In your groups you came up with a starting list of the process. Try to answer the questions below

1. What are the main molecules involved in translation?
2. What is mRNA translated into?
3. What are the building blocks of that molecule?
4. What are codons and anticodons?

If you did not like the crashcourse video here are a couple more!



 (start at 5:15)

Or the HHMI video

Don't forget your homework is to continue the gizmo and read the Timid Mice article (on Veracross)