HW 5-8

Question 1

What is synthetic biology?

Answer 1

a technique used to create genetically engineered organisms.

Question 2

Name one application of synthetic biology

Answer 2

malaria treatment

Question 3

What is DNA scar?

Answer 3

In order to make recombinant DNA we often need to incorporate restriction sequences in the DNA sequence. As a result, when this DNA is replicated, we have a segment of restriction
sequences that we do not want which is called DNA scar.

Question 4

Other than the formation of DNA scars, what is another limitation of constructing a full megabase
size genome using cut and paste?

Answer 4

limited to the number of DNA fragments we can incorporate into the single DNA molecule.

Question 5

The genome of what organism was first synthesized in 2004? What is the size of the DNA molecule
that was synthesized?

Answer 5

The genome of Mycoplasma genitalium was first synthesized in 2004. The size of the DNA molecule is 582,970 bp.

Question 6

How are the homologous “sticky end” sequences created in Gibson Assembly?

Answer 6

Homologous “sticky end” sequences are created in Gibson Assembly by making sure that the
3’ end of one fragment is the same sequence as the 5’ end of the other fragment. This
ensures that the two sequences anneal to each other. As a result of these two sequences
annealing to each it initiates DNA polymerase to start adding nucleotides to create a long
DNA strand.

Question 7

How does Gibson Assembly avoid
the need to use restriction enzymes and the formation of a DNA scar?

Answer 7

This avoids the use of a restriction enzyme and the formation of a DNA scar because there will be no overhanging sequence in the DNA strand.

Question 8

What is the function of DNA Polymerase and DNA ligase in Gibson Assembly?

Answer 8

The function of DNA Polymerase in Gibson Assembly is to add the nucleotides to each of the
strands after the homologous ends have annealed to each other. The function of DNA ligase
in Gibson Assembly is to seal the remaining nicks.

Question 9

How many DNA fragments have been joined together into a single molecule using Gibson
Assembly?

Answer 9

Six DNA fragments have been joined together into a single molecule using Gibson Assembly.

Question 10

What is auxin?

Answer 10

a plant hormone that regulates elongation and division of plant cells.

Question 11

What is cytokinin?

Answer 11

a plant cell hormone that works with auxin to help plant cells divide.

Question 12

What are opines?

Answer 12

is the substance that only the Agrobacteria can use as food.

Question 13

What are crown galls?

Answer 13

a plant tumor that results from Agrobacteria infection due to uncontrolled cell division.

Question 14

How does Agrobacteria genetically modify an infected plant?

Answer 14

Agrobacteria genetically modifies an infected plant because when a plant is wounded the plant releases phenolic compounds such as acetosyringone. These phenolic compounds attract Agrobacteria into the wound. Afterwards, the Agrobacteria produces a protein called pilus through which they insert their DNA into the plant cell. Agrobacteria DNA eventually makes it to the plant cell’s nucleus where the Agrobacteria DNA is inserted into the plant cell’s genome.

Question 15

How does Agrobacteria benefit from this parasitic relationship?

Answer 15

once its gene is inserted into the plant cell it will cause the plant cell to produce opines which the Agrobacteria can use as food and energy.

Question 16

Which Agrobacteria protein forms an excinuclease?

Answer 16

vird1 and vird2 are Agrobacteria proteins that forms an exinuclease

Question 17

How does this excinuclease contribute to the genetic transformation of plants?

Answer 17

Exinuclease contributes to genetic transformation of plants because the vird1 and vird2 proteins form a dimer that bind to the left Border and Right Border sequences. Then the exinuclease cuts the single stranded DNA sequence between the left border and right border sequences. This single stranded DNA goes to the pilus and then into the plant cell. Eventually, the sequence reaches the plant cell’s nucleus where it is replicated into a double stranded
DNA molecule and it is eventually incorporated into the plant cell’s genome.

Question 18

What forms the recognition sequence for this excinuclease?

Answer 18

The Left Border sequence and the Right Border sequence

Question 19

What is the Ti-plasmid?

Answer 19

Ti-plasmid is the tumor inducing plasmid that is needed to infect the plant

Question 20

What types of genes are found on the Ti-plasmid?

Answer 20

Genes that code for a m in o acid derivatives such as opines a re also found on the Ti-plasmid.

Ti-plasmid are genes that code for proteins forming the virulence region which is the region needed in order for the
bacteria to incorporate its DNA into the plant cell.

Question 21

How are the Ti-plasmid and Agrobacteria used to introduce a foreign transgene into the genome of a plant to make a GMO?

Answer 21

genes for opine synthesis, auxin synthesis, cytokin synthesis, and opine metabolism are deleted through the cut and paste method. Then the transgene is inserted between the left border and righ border sequences in the place where the T-DNA was initially present. Then this plasmid is transformed into Agrobacteria cells.
In the future, the Agrobacteria will then be able to infect plant cells.

Question 22

What is the title of the article referenced in the video. What is the title of the research paper on which this homework is based? In what research journal was this paper published?

Answer 22

The title of the article that is referenced in the video is “A gene defect may make rabbits do handstands instead of hop.The title of the research paper on which this homework is based is “A loss of function mutation
in RORB disrupts saltatorial locomotion in rabbits.” The research journal that this paper was published in is PLOS Genetics.

Question 23

What is the name of the animal described in this manuscript?

Answer 23

domesticated rabbits sauteur d’ Alfort.

Question 24

What is Saltatorial locomotion and in what types of organisms is it found?

Answer 24

Saltatorial locomotion is a characteristic type of hopping where the animals have a simultaneous movement of the hindlimbs and a rhythmical movement of the forelimbs. Saltatorial locomotion is found in organisms such as rabbits, hares, kangaroos, and some species of rodents.

Question 25

What is full and abbreviated name of the gene described in this manuscript? What type of protein does this gene encode?

Answer 25

RAR Related orphan ReceptorB (RORB)

RORB protein

Question 26

Where is this protein usually distributed in an organism, such as the one shown in the video from question #1?

Answer 26

regions of the nervous system especially in the spinal cord dorsal horn.

Question 27

Given the location at which the mutation occurs, is this effect on the protein expected?

Answer 27

Given the location at which the mutation occurs, which is the transcription factor of RORB, this effect on the protein is expected because transcription factors play a crucial role
in the regulation of gene expression. If there is a mutation in the transcription factors this will lead to a deficit in mRNA synthesis and protein expression because the transcription factors play a significant role in the regulation of
mRNA synthesis and protein expression. Therefore, the mutation in the RORB transcription factors leads to a deficit in mRNA synthesis and protein expression causing there to be extremely low levels of the RORB protein.

Question 28

What is gene drive?

Answer 28

The mechanism of gene drive involves altering a specific gene in a DNA sequence of an organism.

Question 29

How a gene drive ensures that a gene will always be transmitted to offspring even if one parent does not carry the gene.

Answer 29

the modified DNA insert is inserted on both X-chromosomes of female that is required for fertility. The CRISPR becomes selfish and also inserts itself into non-CRISPR DNA in males.