SOP

Methods
https://scholarship.rice.edu/bitstream/handle/1911/104917/RICE2569.pdf?
sequence=1&isAllowed=y
1.
Plasmid science
- Hemoglobin expressed via rHb0.0 plasmid
o Baxter Hemoglobin Therapudics
o pDLIII13e, based on parent vector pKK223-3
 Tac Promoter
 IPTG (Isopropyl β-D-1-thiogalactopyranoside)
 Metaboliv mimic of allolactose
 Allolactose induces protein expression where the gene is under
the control of the lac operator
o There is a multi-cloning site between HindIII and BamHI
 Multi-cloning site: segment of DNA with restriction sites
 (portion in plasmid)
 The MCS allows for the piece of DNA (mutation) to be inserted
into the plasmid
 HindIII
 DNA restriction enzyme
 Cleaves DNA sequence AAGCTT in presence of cofactor Mg2+
 BamHI
 Restriction endonuclease
 Cleaves DNA
 Inside this cloning site, the alpha and beta hemoglobin subunits are
expressed (expressed in succession)
 Tetracycline resistance gene is used as a regulatory gene
o The subunits are cloned into the rHB0.0 plasmid wth a valine -> methionine
replacement @ first positions of the alpha subunit and beta subunit genes
 This allowed the genes to be expressed in E.Coli
 The rest of the sequences were optimized for E. Coli codon bias
 Codon bias: occurs when mRNA coding for foreign protein differs
from E. Coli’s codon usage
o Codon- series of three nucleotides encoding specific R
group on amino acid
o E. Coli naturally wants to use/express certain codons more
than others
1. Use rHb0.0 wild-type genes as parental templates for complimentary synthetic oligonuc
leotides
a. Oligonucleotide = short DNA / RNA molecule
b. ~33 bases long
c. Desired point mutations
d. Note: maintain proper GC percentage and Tm
i. GC content = % nitrogenous bases in DNA or RNA that are G or C
2. Use PCR to incorporate mutations
a. rHb0.0 DNA is denatured, then the oligonucleotide primers anneal to it
b. High-fidelity DNA polymerase extends and incorporates the mutation into each
nascent strand
i. Nascent strand: short strand of DNA/RNA combined into chimera strand
ii. High Fidelity: high accuracy replication of desired template
c.
3. AFTER temperature cycles: Digestion of Methylated, nonmutated parental DNA
a. Parental DNA- the strand that the copies were made from
b. Performed via addition of restriction enzyme Dpn I
i. Type IIM restriction enzyme, cleaves DNA containing methylated adenine
ii. Basically pulls unnecessary/bad methyl groups off of adenine on the
parent DNA strands
4. Check that the DNA is correct
a. Insert the PCR-grown mutated rHb0.0 DNA and the digested parental DNA into
either:
i. DH5α competent cells (E. Coli)
ii. JM109 competent cells
b. Grow competent cells overnight @ 37 Degrees C on tetracycline treated LB agar
plates
c. Isolate DNA from colonies
d. Confirm DNA via primer extension sequence analysis compared to sequencing
primers designed for full rHb gene
i. Use gel electrophoresis
5. Transform wild type and rHb0.0 into expression cell line, SGE1661 (from
Baxter/Somatogen)
a. SGE1661 allows for more rapid uptake of extracellular heme (variant of JM109 E.
Coli)
b. Necessary equipment
i. Shaking incubator at 37 °C
ii. Stationary incubator at 37 °C
iii. Water bath at 42 °C
iv. Ice bucket filled with ice
v. Microcentrifuge tubes
vi. Sterile spreading device
c. Reagents
i. LB agar plate (with appropriate antibiotic)
ii. LB or SOC media
iii. Competent cells
iv. DNA you'd like to transform
d. Steps for transformation (https://www.addgene.org/protocols/bacterial-
transformation/)
i. Ratio of competent cells to DNA: 50–100 µL to 1-10 ng (1–5 µL of ligation
mixture)
ii. Place LB Agar plates w/tetracycline into incubator at 37 degrees Celsius
to warm up
iii. Mix 1–5 µL of DNA into
e. Spread cells onto LB Agar plates with 15 picograms/ml tetracycline
f. Incubate cells for 12 hours @ 37 degrees C
g. Store cells as necessary at 4 degrees C
6. Scrape off 1 colony of bacteria and inoculate 4ml of LB
 a. incubated at 37 degrees C overnight in shaker/water bath
b.

grow E. Coli cells to exponential curve, then chill them in calcium chloride

heat shock them at 45 for

Molecular cloning a laboratory manual 4th edition UMGC website

Materials
- QuickChange Site-Directed Mutagenesis Kit (Stratagene Inc.)
- rHb0.0 DNA
- oligonucleotide primers w/ desired point mutations
- PCR Machine
o Homemade?
o
- DH5α competent E. Coli cells
- Dpn I enzyme
- Tetracycline treated LB agar plates
- LB Broth