YAC AND BAC

AND SHUTTLE
VECTORS
YACs (Yeast Artificial Chromosomes) and BACs
(Bacterial Artificial Chromosomes) are used to clone large
DNA fragments, such as entire genes or gene clusters, which
are too large for conventional plasmids. Shuttle vectors can
replicate in multiple host organisms, allowing for gene
manipulation in different systems, such as bacteria and yeast,
for various research and biotechnological applications.
 YEAST ARTIFICIAL CHROMOSOME
Yeast artificial chromosomes (YACs) are human-made
chromosomes created from yeast DNA. They are
used to clone large sections of DNA in yeast cells.
YACs allow scientists to isolate and propagate
very large DNA segments in yeast. By inserting
large DNA fragments, scientists can clone and
map these sequences using a method called
chromosome walking. On average, YACs can
clone DNA segments of 200 to 500 kilobases (kb),
but they can also hold up to 1 megabase (Mb) of
DNA.
 STRUCTURE

A yeast artificial chromosome cloning vector consists of

two copies of a yeast telomeric sequence (telomeres
are the sequences at the ends of chromosomes), a
yeast centromere, a yeast ars (an autonomously
replicating sequence where DNA replication begins),
and appropriate selectable markers.
 Construction of YAC Vectors
• Linearization of a circular, DNA plasmid by
restriction digestion with BamHI
• Restriction digestion with EcoRI
• Ligation with the DNA fragment of interest
Some examples of YAC vectors are YAC72
and YAC46.
 WORKING PRINCIPLE OF YAC
• The principle is similar to that for plasmids or
cosmids.
• The experimenter introduces some typical elements
that are necessary for correct replication.
Subsequent rounds of replication produce many • In the case of YACs, the replication origins ,ARS,
copies of the inserted sequence, in a genetic the centromeres and telomeres of the yeast
procedure known as cloning. chromosomes, which must be inserted into the
DNA being cloned.
• The constructs can be transformed in yeast
Spheroplast and are then replicated there.
• In contrast to the vectors, YACs are not circular;
they are made of linear DNA.
 PROCESS FOR YAC

• YAC vector is initially propagated as circular • The linear chromosome is again digested at
plasmid inside bacterial host utilizing a specific site with two arms with different
bacterial ori sequence. selection marker.
• The circular plasmid is cut at a specific site • The genomic insert is then ligated into YAC
using restriction enzymes to generate a linear vector using DNA ligase enzyme.
chromosome with two telomere sites at • The recombinant vectors are transformed
terminals. into yeast cells and screened for the
selection markers to obtain recombinant
colonies.
 ADVANTAGES

YACs have the largest insert capacity among cloning systems.

Yeast expression vectors (YACs, YIPs, YEPs) can express eukaryotic proteins
needing post-translational modifications.
Cloning in yeast ensures stability of unstable or underrepresented sequences
compared to prokaryotic systems.
YACs can be reintroduced into mammalian cells to study gene functions in their
native context.
 USES and DISADVANTAGES

• YACs were first made to study YAC libraries often contain chimeric clones with DNA
chromosome behavior in mitosis and from different genome locations.
meiosis without disturbing natural • YAC clones may have deletions, rearrangements, or
chromosomes. noncontiguous DNA pieces, requiring careful
• YAC contigs have created a framework analysis.
for physical mapping of human, • Cloning efficiency is low, with about 1000 clones per
mouse, and Arabidopsis genomes. microgram of vector and insert DNA.
• YACs are favored for whole genome • YACs are less stable than BACs.
analysis. • Isolating YAC DNA from yeast clones yields a low
amount, often less than 1 μg.
 BAC

● BAC (Bacterial Artificial Chromosome)

vectors are a type of artificial chromosomes
designed to transform into bacteria, especially
E. coli, after cloning large DNA fragments.
The size of the DNA fragment that can be
inserted into a BAC vector is 100-200 kb. The
structure of the BAC vector is based on the
fertility plasmid/F-plasmid.
BAC COMPONENTS

● The common components of a BAC vector are;

● repE – Mediates the assembly of the
replication complex
● parA and parB – For the partitioning of genes
during replication
● Selectable marker – For the selection
of transformants; can be an antibiotic resistance
gene or LacZ
● T7 and SP6 – Promote the transcription of
the insert
● OriS – For the unidirectional origin of replication
CHARACTERSTICS
• The original BAC vector, pBAC108L, is derived from the mini-F plasmid pMB0131,
containing genes for self-replication and copy number regulation.
• Essential genes oris and repE allow self-replication, while para and parB regulate copy
number to one or two per E. coli genome.
• The vector includes multiple cloning sites flanked by T7 and SP6 "universal promoters,"
bordered by GC-rich restriction enzyme sites for insert excision.
• It contains cosN and loxP sites for linearization by bacteriophage I terminase and P1 Cre
recombinase, aiding restriction mapping.
• A chloramphenicol resistance gene enables negative selection of non-transformed
bacteria.
• The vector is 6900 bp long and can hold insert DNA exceeding 300 kilobases (kb).
OTHER BAC VECTORS
GENE CLONING
SHUTTLE VECTORS

Shuttle vectors carry origins of replication from two hosts, allowing them to move between them.
● They can replicate in both mammalian and bacterial cells.
● Shuttle vectors are hybrids with sequences from bacterial plasmids and mammalian viruses.
● They contain three key DNA sequences: a viral replication origin, a bacterial replication origin, and a
drug resistance gene.
● Different replication and repair sequences enable their maintenance in bacterial cells.
● There are three types of shuttle vectors: transiently replicating, episomal, and integrated, each
utilizing different replication systems.
● Transiently replicating vectors require recognition by large T antigen for replication in human cells.
● Episomal vectors establish cell lines that replicate plasmid DNA containing the insert.
● Integrated vectors replicate after fusion with specific cell types for gene expression.
Yep vector

The pJDB219 ia an example of yeast

episomal plasmid it include :-
● 1. The 2 micron plasmid
● 2. LEU 2 gene
● 3. The pBR 322
References

● https://www.sciencedirect.com/topics/neuroscience/yeast- artificial-chromosome
● https://nptel.ac.in/courses/102103013/module1/lec4/11.html
● https://www.ncbi.nlm.nih.gov/pubmed/7859160
● https://www.ncbi.nlm.nih.gov/pubmed/9291964
● https://www.slideshare.net/gurya87/yeast-artificial-chromosomes- yacs-44970900
● https://www.encyclopedia.com/science-and-technology/biology-
01
The role of
science in
everyday life
How present is it in our daily lives?
It’s experiment time!
By conducting experiments, students move beyond
theoretical learning and experience science in action.
Here are three examples for your class:
● The egg drop
● Homemade lava lamp
● Invisible ink with lemon juice

These hands-on activities not only make lessons more

engaging but also foster a deeper understanding of
scientific concepts
Understanding the scientific process

Understanding Applying
The scientific process begins with The application doesn't end at
observing a phenomenon and asking conclusion. If the results support the
questions about it. This leads to hypothesis, further tests might be
formulating a hypothesis, which is an conducted for validation. If they don't,
educated guess that can be tested. After the hypothesis may need to be revised.
conducting experiments to test this The findings are finally shared so they
hypothesis, the collected data is analyzed can be evaluated by the scientific
to draw conclusions community
Some examples of case studies
Effect of light on Bird species
Water quality testing plant growth identification
By testing a local pond or river, This involves growing plants Students count local birds over
they'd learn about indicators of under different light conditions a period of time and identify
water health different species

Invasive species Observing weather

Chemical reactions impact patterns
They will observe the chemical They can research how the Over a period of time, they
reactions that occur while species was introduced and its track weather patterns to
baking effects on the ecosystem understand climate trends
 Every question you
ask is a step towards
knowledge
Visual elements help illustrating your
information
100,000
Did you know that an average human heart beats
around these many times a day?
Some interesting country studies

USA
National human genome research

DRC
Ebola virus disease

New Guinea
Climate studies
The steps for a successful experiment
Think about what you want to Make sure to record all your
discover and guess the outcome observations and results

Define the hypothesis Conduct it and collect data

Analyze results and draw

Plan your experiment conclusions
What materials will you need? What Look at the data you collected. Did it
steps will you follow? support your hypothesis?
What materials did we use?

Wood Plastic
This is a natural This is a synthetic
material material

Resin Brass
This is a composite This is a metallic
material material

Follow the link in the graph to modify its data and then paste the new one here. For more info, click here
Rubric
Criteria Exceptional (4) Proficient (3) Basic (2) Limited (1)

In-depth; accurate; Solid; accurate; clear Basic; somewhat Limited; contains

Scientific content
interconnected examples accurate. Lacks depth inaccuracies

Well-structured; Logically organized; Somewhat organized;

Disorganized; minimal
Critical analysis insightful; exceptional thoughtful analysis; lacks depth; basic
critical analysis
evaluation good critical thinking critical thinking

Clear; coherent; Communicates clearly; Somewhat clear;

Unclear; disorganized;
Clarity and focus maintains focus with generally smooth occasional lack of
lacks coherence
smooth transitions transitions coherence

Integrates varied, Effectively uses Includes some examples;

Application of Lacks sufficient, relevant
relevant examples examples for clarity and limited in number or
examples examples
effectively relevance relevance
Short answer questions
The students should read the following questions and answer them in their paper. These questions
require students to demonstrate their understanding of key concepts

1 What is the scientific method, and why is it important in scientific research?

Describe a natural phenomenon that captivates your curiosity. How would you approach
2 investigating and explaining it scientifically?

3 In what ways do scientific discoveries impact our daily lives?

Group discussion
Divide the class into small groups of 3-5 students. Assign each group a discussion topic or question. In
their groups, they should discuss the topic and reach conclusions

The wonders of space Genetics and heredity

Incredible world of
Topics Ecosystems and biodiversity
microorganisms

The scientific method in

Renewable energy sources
everyday life
Essay
Select a topic for the essay. Students should research the topic,
outline the structure and write a well-constructed essay

Topics
● The interconnectedness of scientific disciplines: Choose two
scientific fields and discuss how discoveries in one field can
influence and contribute to advancements in another
● The role of science in addressing global challenges: analyze
the role of science in addressing global challenges. Choose one
or more pressing issues and discuss how scientific
advancements contribute to understanding, mitigating, or
solving these challenges
Conclusions

Scientific Understanding
method Data representation materials
The process of conducting an Visual representation of data Knowledge about different
experiment is crucial to can improve the understanding materials is fundamental in
understanding science. It helps and interpretation of the science. This understanding can
students learn how to results. It provides a clear way help students comprehend why
hypothesize, plan, execute, and to present complex data sets certain materials are chosen for
analyze data in a systematic and allows for easy specific purposes
way comparisons
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