Unit 1

______ --> DNA ------ --> RNA ------ --> Protein

DNA is replicated by a ___ ________ to make copies of DNA. ___ can be transcribed by an RNA polymerase to make RNA. RNA is then translated by ________ to make protein.

Looking at the anatomy of a gene, there are three parts on the DNA. They work in a line. (1)________(2)CODING SEQUENCE(3)___________

Imagine we are looking at the anatomy of a gene on DNA. Upstream of the gene/coding sequence there is a __________ that recruits ___ polymerase. Downstream of the gene there is a __________ sequence

DNA polymerase uses _____ as template to synthesize a new molecule of ____, where RNA polymerase uses ____ as template to synthesize a new molecule of ___.

In DNA replication: (1) What molecule is the template? (2) What molecule is being synthesized? (3) What are the monomer subunits? (4) What enzyme makes the polymer?

What three components make up a nucleotide/s?

In RNA transcription: (1) What molecule is the template? (2) What molecule is being synthesized? (3) What are the monomer subunits? (4) What enzyme makes the polymer?

In Protein translation: (1) What molecule is the template? (2) What molecule is being synthesized? (3) What are the monomer subunits? (4) What enzyme makes the polymer?

TRUE OR FALSE: The purpose of the promoter is to regulate gene expression and recruit the RNA transcription machinery.

GO TO YOUR CAMERA ROLE AND DRAW OUT LAC OPERON

We will exploit the ___ operon to regulate CaMPARI gene expression

What is CaMPARI?

CaMPARI is a novel engineered _____________ protein. The original sequence came from the lobed _____ coral. It was engineered to be capable of detecting changes in _______ signaling and can be used as a “sensor”. If a molecule of _____(redreen) CaMPARI is bound to Ca2+ and violet light is present, it can undergo an ________(reversable/irreversable) photoconversion to become ______(redreen).

Note that CaMPARI isn’t actually green or red. It fluoresces that color upon applying the excitation and emission wavelengths.

This is an extrachromosomal, circular DNA molecule
-- It carries growth advantages
-- It replicates independently of chromosome
-- It is used as a vector to carry foreign DNA into target cell

In lab, we will create ________ to express CaMPARI. This is our experimental goal for BIT 410/510. We want to create a _______, an extrachromosomal circular piece of DNA that carries the CaMPARI gene sequence

This semester, we will _____ and express CaMPARI in both _________ and ___________.

Prokaryotes and Eukaryotes have fundamental differences.

Who is who?

(1) Extra-chromosomal DNA is replicated and passed on
--- Cheapest, easiest way to replicate a DNA molecule

(2) Extra-chromosomal DNA is usually NOT replicated
--- More expensive to express some protein
--- CAN perform post-translational modifications

Lab goals summed up:

(1) We will begin by _______ CaMPARI from a vector we obtained. We will amplify it using polymerase chain reaction (___).

(2) We will then use a modern cloning technique called Gibson Assembly to assemble the ________ fragment into a ________ expression vector for expression in Chinese Hamster Ovary cells (___ cells, a mammalian cell line).

(3) We will also use a ________ expression vector that allows us to make CaMPARI in Escherichia coli for purification.

(4) We will then screen our clones to make sure they are correct using several approaches: _________ enzyme mapping, PCR screening, and Sanger DNA sequencing.

(5) We will then express and purify our recombinant __________ protein and do some expression studies and Western blots.

(6) Finally, we will use CaMPARI as a _______ indicator in CHO cells as part of the experiment you help design.

Match the vocabulary term with its definition for this course (genetic engineering, biotechnology, molecular cloning)

(1) The use of biological systems and organisms to develop or make useful products

(2) The application of molecular biology methods to manipulate the genome of an organism

(3) The use of methods to produce new recombinant DNA molecules and to direct their replication

Which of the following is NOT an example of biotechnology?

a. Yogurt

b. Using a computer to take lab notes

c. Screening for antibiotics

d. 3D printing skin grafts

Which of the following best describes the traditional central dogma of molecular biology?

a. DNA is transcribed into mRNA which is translated into protein

b. DNA is replicated into mRNA which is transcribed into protein

c. DNA can be produced by replication or reverse transcription

d. DNA is replicated into RNA, some of which is translated into protein

What is the typical flow of the scientific method? [Put in order]

(Determine appropriate experimental controls | Analyze the results | Perform an experiment | Compose a testable hypothesis)

Step 1:

Step 2:

Step 3:

Step 4:

Match the vocabulary term with its definition for this course. (vector, molecular cloning, plasmid, biotechnology)

(1) The use of methods to produce new recombinant DNA molecules and to direct their replication

(2) The use of biological systems and organisms to develop or make useful products

(3) An extrachromosomal, circular DNA molecule

(4) A DNA molecule capable of replication in a host organism, specially modified for propagating DNA sequences or protein expression