BIO 12.

01 NOTES Nomenclature:
11-07-2022
Group Reporting
- Topic – Microbial Biotechnology
- Definition of the topic
- 2-3 examples of what the topic is about (ex.
actual products)
 Talk about the process (how did the scientist
create the product) – do not go to deep,
make others understand it (try to apply the
lecture topic)
- References
- Video should be 15-20 mins long
- Submission will be on the 3rd of November
- Long exam will be on the 28th of November
- Quiz 5 (online) will be done online on the 17 th
of November
Recombinant DNA Technology
- Isolating a gene and placing it in another
organism to improve or create a new product
- Bacteria has a circular DNA
- EcoRI (eco r one) – isolated from e. coli, strain
RY13, enzyme I
- BamHI (ban h one) – isolated from
amyloliquefaciens, strain H, enzyme I
Restriction enzyme
- Blunt ends – cut at the same position
- Sticky end – restriction enzyme cut it at a
different site
- Result in overhangs – expose bases
- Can spontaneously reattach
LIGASE
- Connecting 2 different DNA from 2 different
organisms
- Act as a glue to make sure fragments are
connected
- Can combine from different organisms
- Cuts are complimentary to each other
- DNA ligase will ensure that the gaps/spaces are
sealed
- Once done, recombinant DNA is done

To genetically manipulate DNA DNA Cloning and Cloning Vectors

- Gene must be isolated
- A vector (vehicle) – a carrier of that target gene Steps
- Recombinant DNA molecule - Isolate
- Obtain many copies for further study - Ligating the DNA into a vector (insert into the
- Obtain valuable protein product vector/vehicle)
- Transform the host cell with rDNA
RESTRICTION ENZYMES (scissor) - Selection of host cell
- Removing the target DNA from the original - Screening
source
- Act as scissors that cut both strands of DNA For a successful to happen
- Recognize a specific site in the DNA - Hope it will survive
- Scientist can design the restriction enzyme - Then replicates
- DNA ligases – seal the nicks from combining 2
molecule together Cloning vectors
- Carry the organism
More info on restriction enzymes… - Conditions:
- Found in bacteria  Has an origin of replication (to make sure it
- A defense mechanism for bacteria grows and pass down)
- Bacteria can be infected with viruses  Must have a small size without undergoing
- Bacteriophages - … degradation
- Inactivate the viral DNA to make sure an  Must have several restriction sites (site in
infection doesn’t happen the DNA that targeted)
 Selectable markers – identify if the
Types experiment is a success
- I and III – restriction can cut at a different point
- II – restriction enzyme cut at that exact point
Plasmids – the ones contain genetic information
yet not necessary for survival (antibiotic
resistance, heavy metal resistance)
Bacterial Vectors
pBR322
- Gene map – tells that it is a circular DNA and
has several special area that will encode for
special characteristics
- P – plasmids, BR – Bolivar and Rodrigues, 322
– id no,
- Tet s – succeptible
Why pBR322?
- Has an origin of replication
- Must be small in size (4363 base pairs –
considered small)
- Several restriction sites – bam h one may cut
this out and etc.
- Selectable markers – can see visually to make
sure experiment is a success (the cells has taken
up the plasmids) insertional inactivation –
means that when inserted with something in it
will be inactive; insert something, then it
inactivates the gene
Screening
- Actual technic in the lab to visually observed
the change
- Replica plating – bacterial colonies present in
the master plate then compare
 Observable results – presence/absence of
colonies
- Nutrient aggre on plates – a house set up for
organisms
 Use to grow them
 Colonies growing on the surface on the
medium
 Each colony is a representative of the cell
 Tells that there is a cell and it grows
overtime
Replica Plating
- Used a sterovelvet cloth
- Making an imprint of the colonies then
stamping it in another plate
- Tetracycline susceptible – it will not grow
- Compare the plate to the master plate and look
for colonies that did not grow (it did not grow
with tetracycline)
- Method of screening cells
pUC
- pUC plasmids
- has an origin of replication despite not seen
- 2686 base pairs (smaller)
- Multiple restriction sites
- Selectable markers here has to do with antibiotic
resistance
- Has another selectable marker – for metabolic
reaction
- Selectable marker – can visually see/observe
- Metabolic reaction as basis of the selectable
marker
pUC and a- complementation (alpha)
- If there is a metabolic reaction, there will be a
product
- So that there will be a visual observable
something
- Screening process
- Alpha c. because of a specific part of the gene
that detected
- Quick and will give results in less than 24 hours
- Replica plating have 2 rounds
- Usually for plates/agar media (master plate) –
takes 24 hours for colonies to grow (takes 48
hours more or less in replica plating)
- In pUC, it only takes a day
- lacZ gene – the blue arrow
 capable of producing the enzyme caked
B(beta)-galactosidase (produce of there is a
lacZ gene)
 can be divided into 2; lacZ-alpha and lacZ-
omega
 lacZ alpha (more important) – will produce
the Beta G. as it encodes functionality
(without it, there will be no BG)
- in replica plating, look for the absence or the
presence of colonies
- in a-complementation, there is always a colony
(based on color)
- blue colonies - did not meddle with the plasmids
- white colonies – if the plasmids are meddled
with
Culture media – food for microorganisms
- gelatin-likeke material allows organisms to
grow
- ITPG – an ingredient that organisms utilize if it
can produce the Beta G.
- X-gal – a dye that reacts with the Beta G (results - Electricity – will make the DNA run through the
to the blue color) gel
- Alpha complementation - In agar rose, it is square and has wells
- Vector + insert ligation – white ones are the - DNA is negatively charge which results the
successful cells DNA to run downwards towards the positive
end when electrified
rDNA Techniques - Ethidium Bromine (EtBr) – stain the DNA so
- Make copies of isolated DNA (several) so that if that when looked under UV light, it will floures
it fails, there is backup (through polymerase - If longer DNA, it will travel slower
chain reaction)

Gel Electorophoresis

Polymerase Chain Reaction -

- Put target DNA with something (cocktail mix = W
enzymes and primers) in the machine then gets
billions of copies in an hour
- PCR does replication again and again until there
is enough DNA copies
- What happens inside the machine?
 Change of temperature

- Denauration
 94-96 degrees Celsius
 Allowing more bases to pair with it
 Usually occur within a minute
 The double stranded DNA will separate
 Then it cools down
- Taq polymerase
 Same with DNA polymerase III
 Taq because it is isolated from
Thermophilus aperatious (thrives in high
temeperature)
 Ensures that enzyme are stable despite the
high temperature
- The PCR machine undergoes hundreds of cycles
to produce billions of copies
Gel electrophoresis
- Combines the use of an agar rose and electricity
current
- Plasmids are small base on the number of base
pairs
- DNA band sizes – see certain band with a
specific size
e put ladder/marker/standard in the first well
(usually comes with the kit)
 Contains a guide/pattern to identify
unknown bands of DNA
 The ladder comes with a kit which tells the
sizes of the DNA
 Fragments – broken down DNA with
unknown sizes
SCHEDULE FOR THE END OF THE
SEMESTER:
- November 14 – supplementary videos posting
- November 17 – Quiz #5 on rDNA online
- November 21 – start of reporting videos
submission
- November 28 – 3rd Long Exam

- Gel – made up of agar/agar rose; basically

seaweed Jell-O
 Act as a filter