Quiz #3

Biochemical Engineering

Fall 2003

Instruction

Any act of academic dishonesty will not be tolerated.

1. (50 pts.) Briefly describe/contrast the following concepts/terms.
a. (10 pts.) Replication, Transcription, & Translation

Solution:

Replication: Synthesis of DNA molecule based on matching the

nucleic acid
sequence of the complementary strand (A=T, C-=G).
Transcription: Synthesis of mRNA from a DNA template (a gene, a
segment of
DNA between the promoter sequence and the
terminator sequence).
Translation: Synthesis of peptides/protein molecules based on
the codon
sequence of an mRNA molecule.
b. Operon & Regulon

Solution:

Operon: A set of contiguous genes under the control of a single

promoter-operator. (e.g., couple antibiotic resistence
and the
target product.
Regulon: A repressor/inducer that affects many noncontiguous
gene products
controlled by separate promoters. (e.g., heat shock
proteins.)
c. Sigma/Rho factor

Solution:

Sigma factor helps attaching RNA polymerase to the promoter on DNA (thus,
starts transcription).
Rho factor releases RNA polymerase (thus terminates transcription).

d. Genome/Gene

Solution:
 Genome: The makeup (sequence) of all the DNA in a cell.
Gene: The portion of DNA that encodes for one protein.
e. Exon & Intron

Solution:

Exon: coding segment in DNA that remains in mature mRNA.

Inton: non-coding segment in DNA that is excised out during
post-transcriptional
modification and is absent in mature mRNA.
f. PCR

Solution:

Polymerase Chain Reaction (PCR): A way to multiply the number of DNA copies
by cycling the temperature in the presence of NTP, primers, and DNA
polymerase. (At low T, two primers, which recognizes specific sequences in a
DNA attach to the DNA to mark the segment to be replicated. DNA polymerase
elongates the complementary chain. At high T, complementary DNA strands
separate. The temperature cycle is repeated to double the number of DNA
segments.)

g. RT-PCR

Solution:

Real-Time Polymerase Chain Reaction (RT-PCR): Quantify the amount of DNA

by examining the number of amplification cycles needed to achieve a specific
DNA level.

h. Restriction Endonuclease & Ligase

Solution:

Restriction enzyme: Digests DNA at specific recognition sites

(DNA scissors).
Ligase: Form permanent bonds between 3' and 5' of
two nucleotides
(DNA glue).
i. DNA Microarray & Gene Chip

Solution:

Microarray: many spots of single-stranded cDNA adsorbed on a glass slide. Gene

Chip: many spots of oligomers (15~20-mers of ssDNA) synthesized
photolithographically on a surface.
2. (10 pts.) List the experimental steps to remove a specific segment (say an intron) from a
plasmid.

Solution:

See Homework #7. Briefly, design two primers to flank the segment to be removed and
perform PCR.

3. (40 pts.) Central Dogma of Biology. Attached is the gene sequence file of pBR322.
Features relevant to this problem are marked with a bar. You may directly mark these
pages to answer the following questions.
a. Give the number of each of the bases in this plasmid.
b. Bases Number
c. ---------------
d. A
e. C
f. G
g. T

Solution:

The sequence file gives the following base counts: "983 a; 1210 c; 1134 g; 1034
t". Note that A & T are always present in equal numbers; so are C and G.

-----------------------
Base Count
-----------------------
A 983+1034 = 2017
C 1210+1134 = 2344
G 1134+1210 = 2344
T 1034+983 = 2017
------------------------
total = 8722 bases (or 4361 base pairs, bp)
h. Beta-lactamase is one of the main features of pBR322. What does beta-lactamase
do?

Solution:

Beta-lactamase opens the beta-lactam ring in certain antibiotics, e.g., ampicillin.

Thus, it destroys antibiotics and allows bacteria to grow.

i. Is the given strand a sense strand for tetracycline resistance protein? For beta-
lactamase?
j. tetracycline resistance protein: |Sense|Antisense|Both|Neither|
k. beta-lactamase: |Sense|Antisense|Both|Neither|

Solution: Antisense strand for tetracycline resistance protein and sense strand for
beta-lactamase.
l. Mark on the sequence sheet the portion (or give range of bases) that is transcribed
into mRNA for beta-lactamase.

Solution:

From the given information on codon sequence and ribosome binding site,

CDS complement(3293..4153)
RBS complement(4161..4165)
Promoter complement(4188..4194)

we conclude that the mRNA for beta-lactamase ranges from <3293 to >4165, but
<4188. Note that the promoter sequence is not part of mRNA, but RBS is.

m. How many amino acids are in beta-lactamase as it is synthesized at a ribosome?

Solution:

The "Feature" gives "CDS complement(3293..4153)"

Number of DNA bases: (3293..4153) = (4153-3296+1) = 861
Minus 3 stop codons (because stop codons do not code for any amino
acid): 861-3 = 858
Number of peptides in beta-lactamase = 858/3 = 286 peptides
n. How many amino acids are in an active beta-lactamase?

Solution:

The "Feature" gives "mat_peptide complement(3296..4084)"

Number of peptides: (4084-3296+1)/3 = 263
o. (10pts.) Circle the two codons that correspond to the first two peptides at the
amine terminus of a premitive beta-lactamase. Repeat for the first two peptides at
the acid terminus. Mark the amine and acid ends. Give the two peptides at the
amine terminus and the two peptides at the acid terminus.

Solution:

Relevant "Feature": "CDS complement(3293..4153)

The relevant gene code section:
3241
ttaccaat
3301 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
atagttgcct
:
4141 gttgaatact cat

base number 3293 4153

| |
DNA (given sequence) 5'-...tta cca atg ... act cat...RBS...-3'
DNA (anti-sense) 3'-...aat ggt tac ... tga gta...RBS...-5'
mRNA 3'-...aau ggu uac ... uga gua...RBS...-5'
beta-lactamase COOH------W---H--....-S---M--NH2
beta-lactamase COOH-----Trp-His.....Ser-Met-NH2