Quiz #4

Biochemical Engineering

Fall 2003

Instruction

Any act of academic dishonesty will not be tolerated.

1. (15 pts.) Briefly describe/contrast the following concepts/terms.
a. Gene Therapy

Solution:

Deliver gene needed to produce a therapeutic protein into cells. Rather than
producing the protein in vitro then injecting it into the body, the protein is
produced within the host cells that require treatment. A common delivery vehicle
is an adenovirus.

b. Exponential Growth & Balanced Growth

Solution:

Exponential growth is where cell density increases exponentially with time.

Balanced growth is a condition where all cellular components are increasing at the
same rate, i.e., where the cellular fractions remain constant as cells grow.
Balanced growth is usually achieved during the exponential growth phase.

c. Intrinsic & Extrinsic Variables

Solution:

Intrinsic variables describe the conditions inside the cell. For example, intrinsic
concentrations indicate the amount of chemical compounds per cell volume;
whereas, extrinsic concentrations indicate the amount per reactor volume. Rate
expressions in a structured model must be based on intrinsic variables, because
reactions take place inside the cell.

2. (35 pts.) You wish to insert PEDF into pUC18 to create a recombinant plasmid. The
codons are in capital letters. The sites in PEDF and pUC18 recognized by the restriction
endonuclease Pst I are listed below.
3. Pst I recognizes the sequence ctgca/g
4. PEDF pUC18
5. -----------------
6. 112* 269
7. 1121
8. 1266*

A summary of sites on pUC18 are listed below.

-------------------------------------------------------------
Bases Functions
-------------------------------------------------------------
142- 147 Promoter for beta-gal (-30 signal), induced by IPTG
176- 180 Promoter for beta-gal (-10 signal)
216- 539 beta-galactosidase codons
230- 289 mcs (polylinker)
885-1745 beta-lactamase codons
-------------------------------------------------------------

A codon translation table is also provided.

a. After treating PEDF and pUC18 with Pst I enzyme, what is the size of the desired
plasmid (in terms of bp, base pairs)?

Solution:

Add the size of pUC18 (1..2686) and that of the PEDF gene segment (112..1266)

2686 + 1266-112+1 = 3841 bp

b. What are other possible outcomes of recombination? Give four examples.

Solution:

Three cuts into PEDF generates 4 segments: A, B, C, D. There are an infinite

number of possible outcomes. Among the most common ones are: pUC18
(circular monomer), pUC18 (circular dimer), B (circular monomer of the first half
of PEDF), C (circular monomer of the second half of PEDF), A-B (circular
monomer of PEDF), A-B' (circular monomer of PEDF where A and B segments
runs toward each other), A-B-A-B (circular dimer of PEDF), A-B-B-A, A'-B-A-B,
... , A-C (linear with one sticky end), A-B (linear with one sticky end), A-C-D
(linear), A-B-D (linear) pUC18-B'-A' (PEDF inserted completely backwards into
pUC18), PUC18-B'-A (half of PEDF inserted backwards), ...

c. (15 pts) Mark on the PEDF gene sequence the portion to be lifted out. Mark on
the pUC18 gene sequence (with an arrow or bar) the location of gene insertion.
What is the size (in terms of number of peptides) of the new protein expressed?
Will this protein possess a correct primary structure that includes the original
PEDF?

Solution:
 The insertion point has the following DNA sequence, where the first red "ATG" is
the start codon for the original beta-galactosidase, and the second red "ATG" is
the start codon for PEDF. The codons near the pUC18-PEDF boundary are
identified with green-colored bases.

pU18 1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat

gcagctggca
pU18 61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
tgagttagct
pU18 121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
tgtgtggaat
pU18 181 tgtgagcgga taacaatttc acacaggaaa cagctATGAC CATGATTACG
AATTCGAGCT
pU18 241 CGGTACCCGG GGATCCTCTA GAGTCGACCT GCA
PEDF 61
gcgt
PEDF 121 atccacaggc cccaggATGC AGGCCCTGGT GCTACTCCTC TGCATTGGAG
CCCTCCTCGG
PEDF 181 GCACAGCAGC TGCCAGAACC CTGCCAGCCC CCCGGAGGAG GGCTCCCCAG
ACCCCGACAG
PEDF 241 CACAGGGGCG CTGGTGGAGG AGGAGGATCC TTTCTTCAAA GTCCCCGTGA
ACAAGCTGGC
:
... continued

Preceeding the start codon "ATG" in PEDF, there are 78 bases:

# bases from pUC18 (216..273) = 58

# bases from PEDF (117..136) = 20
--------------------------------
# bases in extra leader = 58+20 = 78 bases
# peptides in leader = 78/3 = 26 peptides

Since 78/3 has no remainder, there is no frame-shift problem and the resulting
protein product completely encompasses PEDF. This can also be deduced visually
from the above base sequence by grouping codons of 3 bases each.

bases of PEDF (137..1222) = 1086

# peptides in original PEDF = 1086/3=362
# peptides in leader = 26
----------------------------------------
# peptides expressed in recombined pUC18+PEDF = 26+362 = 388
d. In case the last part does not work, what do you propose?

Solution:

Try another plasmid, including pUC18's brother, pUC19. Or try a different

restriction endonuclease.

e. If you insert the above gene product into E. coli by transforming it, outline a
screening method to isolate the ones that produce PEDF.
 Solution:

Plate transformed cells on a petri dish containing ampicillin, X-gal, and IPTG.
Pick out white colonies.

9. (20 pts.) Draw a typical cell growth curve for a batch fermentation. Label the major
phases. Briefly describe what is occurring in each phase.

Solution:

| ..........
| . .
| . .
Cell Density | . .
| . .
| . .
|.... .
+--------------------------
Time

Major Phase:
1. Lag Phase ... get cell machinery (induction & enzymes)
ready for growth
2. Exponential Phase ... cell growth
3. Deceleration Phase ... effect of substrate depletion, toxin
accumulation, or crowding becomes apparent
4. Stationary Phase ... growth stops
5. Death/Decline Phases ... death sets in
10. (30 pts.) A product is formed with the aid of a microorganism in a bioreactor. Cell growth
is enhanced at low limiting substrate concentration (S) but is inhibited at high substrate
concentration. Cell growth, cell maintenance, and product formation all consume a
limiting substrate. Product (P) forms as a result of both cell growth and cell maintenance.
a. Write dynamic equations that govern cell (X), substrate (S), and product (P) in a
batch bioreactor.

Solution:

dX/dt = μ*X
dS/dt = -μ*X/Yx - m*X - (α*μ*X+β*X)/Yp
dP/dt = α*μ*X + β*X
where μ(S)=μmax*S/(K+S+KI*S*S)
b. Repeat Part a) for a continuous bioreactor.

Solution:

Add the dilution|flow term to the batch equations

dX/dt = -D*x + μ*X

dS/dt = D*(Sf-S) - μ*X/Yx - m*X - (α*μ*X+β*X)/Yp
dP/dt = -D*P + α*μ*X + β*X
where D=F/V
c. Repeat Part a) for a fed-batch bioreactor.

Solution:

Same as in continuous bioreactor, except that the bioreactor volume is changing

with time.

dX/dt = -D*x + μ*X

dS/dt = D*(Sf-S) - μ*X/Yx - m*X - (α*μ*X+β*X)/Yp
dP/dt = -D*P + α*μ*X + β*X
dV/dt = F
where D=F/V
d. Identify the operating parameters (i.e., the variables that an operator has control
over) each of the operating modes.
e. Batch:
f. Continuous:
g. Fed-Batch:

Solution:

Batch: harvest time, initial composition

Continuous: F & Sf (usually kept constant)
Fed-Batch: F & Sf (usually time-varying), harvest time