Unit 11: Molecular Biology II: How Proteins Are Made
Reading
OpenStax book: Ch. 9 - "Molecular Biology" sections 9.3 through 9.5
BJU: 4B - "Cell Metabolism and Protein Synthesis"
AP: Green book Ch. 15 "Genes and Proteins" and Ch. 16 "Gene Expression"
AP students additional reading: Princeton Review "Gene Expression and Regulation" from Central Dogma up to Biotechnology
Topics
Lab
Others:
OpenStax book: Ch. 9 - "Molecular Biology" sections 9.3 through 9.5
BJU: 4B - "Cell Metabolism and Protein Synthesis"
AP: Green book Ch. 15 "Genes and Proteins" and Ch. 16 "Gene Expression"
AP students additional reading: Princeton Review "Gene Expression and Regulation" from Central Dogma up to Biotechnology
Topics
- Week 1: Transcription & translation
- Week 2: Gene expression and regulation
Lab
- Bio-Rad pGLO Bacterial Transformation lab (fulfills AP "dirty dozen lab" # 8).
- PhET Gene Expression lab
- PhET Gene Machine Lac Operon lab
Others:
- HHMI Transgenic Fly lab (fulfills AP "dirty dozen lab" #8)
- CRISPR labs (various sources)
Review
All living organisms are made from four basic molecules - proteins, carbohydrates, lipids, and nucleic acids.
Proteins are long chains of amino acids which are linked together and very carefully folded-up into a predefined shape by molecular machines within the cell. You have approximately 100,000 different kinds of protein molecules - and new ones are being discovered all the time. Proteins are the "workhorse of the cell". They serve as the signaling molecules, enzymes, structural components, docking stations, pumps, and the machinery of the cell. As you might have guessed, proteins are manufactured in a highly-mechanized and information-rich process which rivals a modern automobile factory. When you begin to grasp that, it makes biology and biotechnology leap out at you!
All living organisms are made from four basic molecules - proteins, carbohydrates, lipids, and nucleic acids.
Proteins are long chains of amino acids which are linked together and very carefully folded-up into a predefined shape by molecular machines within the cell. You have approximately 100,000 different kinds of protein molecules - and new ones are being discovered all the time. Proteins are the "workhorse of the cell". They serve as the signaling molecules, enzymes, structural components, docking stations, pumps, and the machinery of the cell. As you might have guessed, proteins are manufactured in a highly-mechanized and information-rich process which rivals a modern automobile factory. When you begin to grasp that, it makes biology and biotechnology leap out at you!
Lecture summary
The Central Doctrine of Biology
The Central Doctrine of Biology
- The central doctrine says, "DNA makes RNA, RNA makes proteins, and proteins make you"
- This is partly true.... your DNA contains the instructions for at least the 'base set' of proteins. However, we have known for at least 30 years that the Body Plan of organisms is not solely determined by DNA, and the carbohydrate signaling molecules contained in the cell membrane contain more information than proteins, and even most RNA goes through a lot of post-transcriptional editing independent of DNA. Therefore, it's not completely accurate to say "proteins make you".
- As a human, you also have a mind - that is to say, you have volition and a will. In other words, you are not just a machine made out of meat. You are also a product of your will and your decisions. You can make good and bad decisions that will affect your life; thus, you are responsible for what happens in many situations. Blaming your DNA for everything is called "Environmental Determinism" - and to be happy and fulfilled in life you want to try to avoid doing that.
- That said, the Central Doctrine of Biology is a pretty good way to memorize the flow of information in protein synthesis.
- This is the process of copying DNA to make mRNA (messenger-RNA).
- When you transcribe something, you copy it. A "scribe" is someone who makes copies of things. Thus, transcription is copying the DNA to make a strand of RNA.
- This is where the mRNA travels outside the nucleus and assembles specific amino acids into specific proteins. The mRNA is like a Post-It Note, carrying the genetic instructions out of the nucleus (the central office) into the endoplasmic reticulum (the assembly area); finding a ribosome (a work station), loading itself into the infeed slot (like a tape reader), being read - 3 bases at a time - by the ribosome, matching-up a tRNA with every 3-base codon (like a factory template), having a specific amino acid attached to the other end of each tRNA in the lineup (like a bunch of subassemblies attached to robot arms), resulting in a string of amino acids becoming attached to one another in a predefined sequence, and that string of amino acids (called a polypeptide at this point) gradually folding-up into a very specific predefined shape, and then moving off to a 2-part folding machine (known as a chaperonin), wherein it is carefully folded into its final shape necessary for whatever purpose the protein serves.
- When you translate something, you write it in a different language. The language of mRNA uses a 4-letter alphabet (the A-U-C-G bases we have been talking about), while the language of proteins uses a 20-letter alphabet (the 20 amino acids we have talked about). Thus, it literally is translating the genetic code from one language into another!
- Thus, the tRNA (transfer RNA) serves as the "dictionary", or Rosetta Stone. If you don't know what the Rosetta Stone was, look it up!... it is a fascinating story in its own right.
- Don't read this! This is only for the enlightened students: if you think about it for awhile, you begin to realize that the information is primary in this whole, fascinating process. It doesn't matter what group of molecules is being used; the information stays primary! Here's an example: I can think of an idea in my mind, write it down on a piece of paper, type it into my laptop, hit a button and send it via radio signals to a box on the wall, the box sends it as pulses down a cable, a radio tower down the street sends it using radio frequency, and someone on the receiving end then goes through the exact process in reverse. In this example, I have used many different mediums to express and store information - but the information is primary! The exact same thing is going on with information in the cell. This has deep philosophical implications, as scientists have known for a long time.
- A gene is simply a section of DNA which encodes for a protein. This is important, and puzzles many students. A gene isn't a "compartment in the cell"! It's just the term we use for a protein-encoding section of DNA... it may be 200 base pairs long, or may be thousands of base pairs long.
- You have around 25,000 genes located on your 46 chromosomes. Mapping various genes and figuring out what they do is the subject of much research!
- These do most of the work in the cell; they form structural elements, signaling molecules, enzymes, and complex machinery of the cell.
- We keep discovering new proteins all the time. You may have as many as 200,000 different types of proteins (the number keeps getting bigger as new proteins are discovered).
- When a newly-manufactured RNA emerges, it is edited to remove "introns" (non-coding regions that you don't want) and keep "exons" (coding regions that you do want).
- Memory device: EXons are EXpressed, and INtrons are IN-between.
- The way the RNA is edited determines what protein it ends up making.
- Because of RNA splicing/editing, each gene is able to make more than one protein! So we no longer say, "One gene, one protein".
| Lecture Slides: Protein Synthesis |
| 11._gene_regulation_slides.ppt |
Video clips below: We will watch and discuss in class
Homework
| 11._gene_expression__homework_questions__rev_2021_.docx |
Genetic Disorder class presentations
"Research a genetic disorder of your choosing, what causes it genetically, and any drugs, procedures or devices used to treat or manage it".
Using this topic, create a PowerPoint presentation with around 6-10 slides, and be prepared to present it at a future class in a 4-5 minute time period. Practice your presentation so it falls within this time period. Upload your completed PowerPoint to Canvas by the due date.
Pre-assignment: You will be asked to submit your "idea" for approval, a week or two before your PowerPoint is due.
pGLO Bacterial Transformation lab
In this 2-part lab we will insert foreign DNA from a glowing jellyfish into some bacteria, and use the bacteria's genetic machinery to express the foreign DNA. The bacteria will then glow bright green under UV light. This is the same technique used in Biomedical Research to make modern drugs
Objectives: learn the techniques of recombinant DNA and gene splicing
In this 2-part lab we will insert foreign DNA from a glowing jellyfish into some bacteria, and use the bacteria's genetic machinery to express the foreign DNA. The bacteria will then glow bright green under UV light. This is the same technique used in Biomedical Research to make modern drugs
Objectives: learn the techniques of recombinant DNA and gene splicing
| pGLO Bacterial Transformation lab__Student_Handout.pdf |
| pGLO_lab__intro PowerPoint |
Background to the pGLO/molecular cloning lab:
- RECOMBINANT-DNA is where we take a piece of DNA from one organism and introduce it into the DNA of another host organism. In this case, we are taking a piece of jellyfish DNA and inserting it into the DNA of a bacteria. The bacteria serves as the HOST organism. The piece of jellyfish DNA is called the GENE-OF-INTEREST. In this case, the section of jellyfish DNA is comprised of a GENE which encodes for a PROTEIN in the jellyfish which causes it to GLOW GREEN in its natural environment (the ocean). The jellyfish manufactures this "green" protein perhaps to defend against predators, or maybe to attract things - we don't really care for purposes of this lab. What we care about is using the MOLECULAR MACHINERY of the bacteria to start cranking-out foreign proteins which could be an important BIOLOGIC DRUG we are trying to mass-produce, let's say.
- GENE SPLICING is the process of taking carefully-chosen molecular scissors - or RESTRICTION ENZYMES - and cutting out the jellyfish "glowing protein" gene and inserting it exactly where we want it on the bacterial DNA. For our lab, the Bio-Rad company has already done this for us. In a real project, you the Bioengineer would select an appropriate restriction enzyme to use as the "molecular scissors" based upon the gene map of the DNA VECTOR you were using
- MOLECULAR CLONING describes the whole process of using E. Coli bacteria as your "host" factory, to manufacture millions & millions of copies (CLONES=COPIES) of your gene-of-interest, and then to crank out large quantities of the protein you are trying to manufacture. Bacteria makes a good factory because it replicates very fast - every 20 minutes.
- This lab exercise illustrates how modern Biopharmaceuticals are made! Instead of making lots of green protein which is a really cool trick, you the Bioengineer would be making lots and lots of Insulin, or Monoclonal Antibodies, or some other biologic drug in order to cure diseases and improve the lives of millions of people.
Lab report
Write a 2 page lab report on the pGLO lab, using 1.2 spacing. Submit a professional-looking report with an appropriate title header, name, date, etc. Address the following questions:
1. What is molecular cloning?
2. What are we doing in this lab? Don't try to outline the whole procedure in your report - I just want to know "what we are doing". What are we trying to accomplish in your own words?
3. We labeled 4 plates as follows: 1) LB, -pGLO, 2) LB, ampicillin, -pGLO, 3) LB, ampicillin, +pGLO, and 4) LB, ampicillin, arabinose, +pGLO. Question: What ends up growing on each plate? Describe what should appear on each plate after incubation.
4. What is the purpose of the LB broth?
5. Why do we use ampicillin in the experiment?
6. What is the purpose of the arabinose sugar in the experiment?
7. What is meant by a "plasmid vector"?
8. The bottom line: why does this bacteria glow green? Explain this in "biology" terms.
9. List 4 or 5 common Biopharmaceuticals or "Biologic drugs" which are produced in this manner, and what do they do? Hint: search "Biologic drugs list" for starters.
Write a 2 page lab report on the pGLO lab, using 1.2 spacing. Submit a professional-looking report with an appropriate title header, name, date, etc. Address the following questions:
1. What is molecular cloning?
2. What are we doing in this lab? Don't try to outline the whole procedure in your report - I just want to know "what we are doing". What are we trying to accomplish in your own words?
3. We labeled 4 plates as follows: 1) LB, -pGLO, 2) LB, ampicillin, -pGLO, 3) LB, ampicillin, +pGLO, and 4) LB, ampicillin, arabinose, +pGLO. Question: What ends up growing on each plate? Describe what should appear on each plate after incubation.
4. What is the purpose of the LB broth?
5. Why do we use ampicillin in the experiment?
6. What is the purpose of the arabinose sugar in the experiment?
7. What is meant by a "plasmid vector"?
8. The bottom line: why does this bacteria glow green? Explain this in "biology" terms.
9. List 4 or 5 common Biopharmaceuticals or "Biologic drugs" which are produced in this manner, and what do they do? Hint: search "Biologic drugs list" for starters.
