Remake of Yeast plasmids & Artificial Chromosomes (G1) by Víctor Manuel Rodríguez García

1. Characteristics (from the presentation and other sources)

vicbios1 — 2021-03-25 16:52:11 UTC

2. Example (diagrams and biobricks)

vicbios1 — 2021-03-25 16:52:20 UTC

3. Application (one example where this vector is used in GE)

vicbios1 — 2021-03-25 16:52:29 UTC

4. References from websites and reviewed sources

vicbios1 — 2021-03-25 16:52:39 UTC

Characteristics

a01704034 — 2021-03-25 17:03:04 UTC

-Replicates only once during the cell cycle
-Contain sequences from around the centromeric region of the chromosomes
-The ARS and CEN are sequences that regulate replication Rodriguez,V (2021)

YIPlac204TKC-GFP-HDEL

a01274235 — 2021-03-25 17:03:31 UTC

Selectable markers TRP1.

2. Example: YEp181-CUP1-His-Smt3

a01208463 — 2021-03-25 17:03:35 UTC

Ori
Promoter and Terminator
Antibiotic Resistance: AMPr
Selectable Marker: LEU2

YRps

a01700974 — 2021-03-25 17:03:51 UTC

Yeast Replicative plasmids
Not looking for recombination
Autonomous replicating sequences
AII ARS (Autonomous Replication Sequences).
Less Efficient than YEps
Requires Constant Selection
-Has a Bacterial Plasmid Origin
-3-100 copies per cell
-Rarely used now

1. Natural occurring plasmids (2µm), 6.3 Kbp

2021-03-25 17:05:57 UTC

It might contain bacterial plasmid sequences
It is the most used
It can integrste into a yeast chromosome (YEp13)
Maria Daniela Delgado

Characteristics Yeps

a01274194 — 2021-03-25 17:06:19 UTC

Replicates autonomously using 2µ origin, high copy number, stable plasmid.

References

a01274235 — 2021-03-25 17:07:05 UTC

https://www.addgene.org/38771/

YIplac204-TC-OM45-FKBPx4

a01422197 — 2021-03-25 17:07:36 UTC

Integrating plasmid for overexpression of OM45-FKBPx4.

Characteristic:
These types of plasmids are the most commonly used when the objective is to have a transgenic yeast.

Application

a01748771 — 2021-03-25 17:07:55 UTC

Overexpression studies where high amount of recombinant proteins are essencial.

YCps - Example

dariolv00 — 2021-03-25 17:08:41 UTC

Ori
lac promoter
AmpR promoter
CEN/ARS
AMPr

Example (diagram and biobricks)

a01376501 — 2021-03-25 17:09:02 UTC

Example YRp (YRp7)

Charateristics

a01209117 — 2021-03-25 17:10:34 UTC

- Without an origin of replication, it needs to integrate to make the replication
-Integrate by recombination
-Function as selective marker

Rodríguez, V., 2021. Vectors In Eukaryotic Cells Genetic Engineering. [ebook] Available at: [Accessed 25 March 2021].

Must be integrated directly into the host chromosome via homologous recombination. -Alba

Aplication

avlz1125 — 2021-03-25 17:10:54 UTC

In most of the cases this plasmids are vectors that are unabled to replicate in the destinantion host.
Thats why are commonly used to achieve target-specific insertion of a construct into a cyanobacterial genome

YEps - Team 2

egruiz0511 — 2021-03-25 17:11:01 UTC

YEplac181-TAF14

Ori
Antibiotic resistece

TFIIS promotes the intrinsic ability of RNA polymerase II to cleave the 3′-end of the newly synthesized RNA.

https://www.addgene.org/8625/

Characteristic

a01376501 — 2021-03-25 17:13:15 UTC

Tend to be unstable and can be lost during budding

1. generated by the insertion of bacterial plasmid sequences and appropriate selection markers

a01274235 — 2021-03-25 17:13:49 UTC

1. bear two characteristic sequences: autonomously replicating sequences (ARS) and centromeric (CEN) sequences

a01274235 — 2021-03-25 17:15:59 UTC

Application:

a01701360 — 2021-03-25 17:16:10 UTC

-Genetic modification of industrial strains of Saccharomyces cerevisiae : lactose assimilation, not harmful genes, food level-grade

Characteristics

2021-03-25 17:16:47 UTC

They are vectors that are unable to replicate in the destination host . This kind of vector can be efficiently transferred into the recipient may be used. QUETA

Applications

a01208463 — 2021-03-25 17:17:22 UTC

Add stability to the hybrid plasmids of E.coli, B. substilis and S. cereviasiae that replicate autonomously, by being used as shuttle vectors.

Applications YRps

a01274194 — 2021-03-25 17:17:33 UTC

Budding yeast S. cervisiae

Copies in YCps

a01209117 — 2021-03-25 17:18:15 UTC

These are considered low copy vectors and incorporate part of an autonomously replicating sequence (ARS) with part of a centromere sequence (CEN).
The copy number of yeast centromeric plasmids is controlled by both the ARS and CEN sequences.
Patrick, M., 2014. Plasmids 101: Yeast Vectors. [online] Blog.addgene.org. Available at: [Accessed 25 March 2021].

Application

a01748771 — 2021-03-25 17:19:36 UTC

Studying effects on a single gene copy

Types of integrative plasmids--

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Queta

Applications

a01748771 — 2021-03-25 17:22:59 UTC

Stable gene integration into the genome or to replace a host gene with another gene or mutant.

YRps

a01209117 — 2021-03-25 17:23:07 UTC

These vectors contain an Autonomously Replicating Sequence (ARS) from the yeast chromosome. These vectors can replicate independently of the yeast chromosome; however, they tend to be unstable.

Patrick, M., 2014. Plasmids 101: Yeast Vectors. [online] Blog.addgene.org. Available at: [Accessed 25 March 2021].

Copies in YEps

a01209117 — 2021-03-25 17:24:52 UTC

These are most similar to bacterial plasmids and are considered “high copy”. The copy number of these vectors can also be controlled if specific regulatable elements are included.
Patrick, M., 2014. Plasmids 101: Yeast Vectors. [online] Blog.addgene.org. Available at: [Accessed 25 March 2021].

Queta

2021-03-25 17:28:13 UTC

https://www.sciencedirect.com/topics/neuroscience/integrative-plasmid

2021-03-25 17:28:47 UTC

QUeta references

a01700974 — 2021-03-25 17:34:43 UTC

HaloTag-bActinCDS-bActin3'UTR-MS2V5

a01274235 — 2021-04-05 16:27:49 UTC

Bacterial Resistance(s): Ampicillin.
Growth Temperature37°C
Copy number: Low Copy

2. pYAC example

a01376501 — 2021-04-06 01:42:31 UTC

pYAC

3. Applications

a01376501 — 2021-04-06 01:45:19 UTC

In which cases the genes are to large to be cloned in procaryotic systems, YACs can be used due to its increased site capacity.

Characteristics

a01209117 — 2021-04-06 16:59:34 UTC

- It is a DNA molecule used to clone DNA sequences in bacterial cells

-Based on the F plasmid
-It has the potential to clone large DNA fragments
-Inserts of 300 Kb
-Low copy number
-Since BACs are much smaller than the endogenous bacterial chromosome, it is easy to purify BAC DNA

References:
-NIH. n.d. Cromosoma artificial bacteriano (BAC) | NHGRI. [online] Available at: [Accessed 6 April 2021].

Rodríguez, V., 2021. Vectors In Eukaryotic Cells Genetic Engineering. [ebook] Available at: [Accessed 6 April 2021].

Application

a01209117 — 2021-04-06 18:06:10 UTC

BACs are used in several applications, from genome sequencing to gene discovery. BACs are the principal driving force to sequence the human genome. For sequencing human chromosome 21 and 22 have been used BAC sub-libraries, since the short DNA insert sizes (100-300kb) and structural stability make BACs the ideal vector for direct
sequencing.
Shizuya, H., 2001. The development and applications of the bacterial artificial chromosome cloning system. [online] Beckman Institute. Available at: [Accessed 6 April 2021].

Characteristics

a01209117 — 2021-04-06 18:23:31 UTC

-It is a modified DNA molecule used to clone DNA sequences in yeast cells.
-YACs are frequently used in DNA mapping and sequencing of genomes.
-Segments of DNA up to one million bp can be inserted into YACs.
-It is based on the YCps (replicative yeast plasmids )but with telomeric ends
- It has the potential to clone large DNA fragments
-Inserts of 100 Kbp to 1.4 Mbp
-Unstable system

References:
-NIH. n.d. Yeast Artificial Chromosome (YAC). [online] Available at: [Accessed 6 April 2021].
-Rodríguez, V., 2021. Vectors In Eukaryotic Cells Genetic Engineering. [ebook] Available at: [Accessed 6 April 2021].

Also, BACs are preferred over YACs because they are more stable and have less chances of having a "chimeric effect" (in some cases as high as 50% of chimerism). Lu X.-H.BAC to Degeneration. Bacterial Artificial Chromosome (BAC)-Mediated Transgenesis for Modeling Basal Ganglia Neurodegenerative DisordersInternational Review of Neurobiology, Volume 89, 2009

a01700974 — 2021-04-06 22:10:20 UTC

-Team 1

Application of BACs

a01274194 — 2021-04-07 23:29:19 UTC

Team 3

- Produce transgenic cells and animals for functional studies of genes
- Functional for gene therapy
- Optimal for long fragments of Genomic DNA or large cDNA up to 300 kb in size
- Made genomic fragments from humans, mice and other species
- Used for transgenesis in higher model cells and organisms

Reference:
Yamaguchi S, Niwa R. (2011) Application of Bacterial Artificial Chromosome Modification System for a Human Artificial Chromosome Vector. [Online] Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3763799/ [Accessed: 7 April 2021].

Example of integrative plasmid

a01704034 — 2021-04-08 00:02:43 UTC

Streptomyces coelicolor example
-Team 1

References:

a01704034 — 2021-04-08 00:04:19 UTC

Heidorn T, Lindbland P. (2011) Integrative Plasmid [Online] Available from: https://www.sciencedirect.com/topics/neuroscience/integrative-plasmid [Accessed: 7 April 2021]

pBacPAK8

a01208463 — 2021-04-08 01:47:10 UTC

Team 3
- Bacterial Resistance (s): Ampicillin
- Growth Temperature: 37C
-Copy Number: High Copy

Reference:
pBacT7-S1T3D (Plasmid #33282)

https://www.addgene.org/33282/

pSmartBAC-S

egruiz0511 — 2021-04-08 16:50:14 UTC

(Empty Backbone) Broad host range bacterial artificial chromosome vector
We first restriction digested pSmartBAC (EU101022.1) with ApaI, end-repaired the DNA to make it blunt and dephosphorylated the digested pSmartBAC vector ends. A synthesized and blunt-ended gene cassette that contained the ɸC31 integrase (int), attachment site (attP) and aac3(IV) that encodes for apramycin-resistance (4,793bp) was ligated to the linearized pSmartBAC-S and apramycin-resistant transformants in E. coli 10G were selected.
Reference:

pSmartBAC-S
(Plasmid #107268)

https://www.addgene.org/107268/

pLV-SI-112

egruiz0511 — 2021-04-08 16:54:30 UTC

Bacterial Resistance(s) Ampicillin
Growth Temperature 37°C
Growth Strain(s) NEB Stable
Copy number High Copy

Target-ACE, which integrates the abilities of both of the previously developed C-to-T and A-to-G base editors by fusing an activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. In mammalian cells, Target-ACE enabled heterologous editing of multiple bases in a small sequence window of target sites with increased efficiency compared with a mixture of two relevant base editor enzymes, each of which may block the same target DNA molecule from the other.

References

pLV-SI-112
(Plasmid #131127)

https://www.addgene.org/131127/

Example-Application

a01376501 — 2021-04-08 20:45:15 UTC

pBS-TarHom-Tol2-kanaR-cryaa-dsRed

"[] allow for the estimation of the endogenous gene expression levels" (Fuentes, F. et al. 2016)
This BAC was developed in zebra fsih and allows the expression od diferent fluorescent proteins

3. Applications

a01274235 — 2021-04-09 01:29:56 UTC

In the actuality they don't have a very important used, but historically they help scientist handle big pieces of DNA, allowing to isolate and reproduce them, as well as isolation of disease genes.

-Originally constructed in order to study chromosome behavior
- Provide physical map framework for the human, mouse, and even Arabidopsis genomes.

References:
Aryal, S. (2019)

Yeast Artificial Chromosomes (YACs) [online] Microbe notes Available at: https://microbenotes.com/yeast-artificial-chromosomes-yacs/ [Accessed 8 April 2021]

NIH. n.d.

Cromosoma artificial de levadura (YAC) [online] Microbe notes Available at: https://www.genome.gov/es/genetics-glossary/Cromosoma-artificial-de-levadura [Accessed 8 April 2021]

pDY-HA1-HA2-neo

2021-04-09 02:30:06 UTC

This is a provider to introduce FR cassette to a BAC by recombineering
Gene name: HA1
Insert size: 527 bp
Species: B. subtilis
pDY-HA1-HA2-neo was a gift from David Gilbert (Addgene plasmid # 109123 ; http://n2t.net/addgene:109123 ; RRID:Addgene_109123)
Team 1

BACs - Example

dariolv00 — 2021-04-09 02:56:38 UTC

*Team 2

MTK678_002

With the purpose to "encode the TU GFP dropout cassette with Ampicillin resistance and bacterial artificial chromosome as a type 678 part to be used in the MTK system".

A Mammalian Toolkit for assembly of large DNA vectors in mammalian cells

Applications

a01748771 — 2021-04-09 03:34:27 UTC

Genome sequencing: BACs are currently being used in large-scale projects to sequence the human and mouse genomes.
BAC contig assembly : The assembly of BAC contigs remains a critical step in the formation of physical maps.
Positional cloning: BACs have been widely used to identify disease loci as well as individual genes
BAC library screening : Another method of using BACs to identify genes entails screening BAC libraries for known sequences.
BAC transgenic mice : The disadvantage of using single gene copies is that oftentimes upstream and downstream elements of the gene are not included in the transgene. An approach to this problem is to introduce transgenes that incorporate. BACs rather than coding segments
BAC microarrays : it will be possible to link gene expression profiles with chromosomal data and DNA sequences in order to achieve a global un derstanding of a disorder or genetic abnormality
https://pubmed.ncbi.nlm.nih.gov/11296661/

Characteristics

a01701360 — 2021-04-09 03:53:18 UTC

Based on the F plasmid wich increases capacity than basic plasmid, the problems of YAC can be solved by BAC also large capacity. (Desmond, 2008)
Large enough to contain a complete gene and its elements (Lai, 2016).
Tool to manipulate gene expression by reporter genes like cells in nervous system (Lai, 2016)
used as promoters of specific genes (Lai, 2016)

Lai, C., 2016. BAC Transgenesis: Cell-Type Specific Expression in the Nervous System☆. Reference Module in Biomedical Sciences, [online] Available at: [Accessed 8 April 2021].

Alejandro Velázquez Velázquez team 3

avlz1125 — 2021-04-09 04:05:05 UTC

Aplication-Example (37161)
Production system for proteín acumulation
This ocures by the use of the pux2410 plasmid in the host train uq4803.
This occurs because the protein contain a igh level of heme cofactor and a low level of FeII-Co.
This cultivation grows quickly and need a temperature of 28 degrees to do it weel.

References
1.- Media.addgene.org. 2021. [online] Available at: [Accessed 9 April 2021].
2.- Addgene.org. 2021. Addgene: UQ4884. [online] Available at: [Accessed 9 April 2021].

Alejandro Velázquez Velázquez team 3

avlz1125 — 2021-04-09 04:12:32 UTC

Example- Aplication (CRISPR-Plasmids)
Base editors
This was crated by fusing Cas9 to to a cytidine deaminase like APOBEC.
They can convert cytidine to uridine with a small editing window; and the the uridine is converted to a thimidine creating a C to T change or a G to A change.
References
1.- Addgene.org. 2021. Addgene: CRISPR Plasmids - Base Edit. [online] Available at: [Accessed 9 April 2021].

applications

a01748771 — 2021-04-09 04:16:45 UTC

-YAC cloning plays an important role in the analysis of a large variety of complex genomes, including those of insects reptiles, birds, mammals and plants
- Contiguous overlapping YAC clones (contigs) form the physical framework of chromosome maps in preparation for large-scale cloning and sequencing of the human genome and the genomes of model organisms.
- YACs are also important for their ability to clone the complete sequences of large genes or gene complexes that exceed the size limit for cloning in conventional bacterial cloning vectors such as plasmids
https://pubmed.ncbi.nlm.nih.gov/7859160/
team 3

Applications

a01701360 — 2021-04-09 04:40:58 UTC

Used for the construction of plant genomes, like the BAC generated libraries for the potato genome for cultivars, breed lines and wild species.
Can be used to isolate genes against common pathogens in potato crops with a gene cloning approach (Glenn, 2007)
Bryan, G., 2007. Genomics. Potato Biology and Biotechnology, pp.179-202.

characteristics

a01748771 — 2021-04-09 04:46:36 UTC

- Plasmid shuttle vectors capable of replicating and being selected in common bacterial hosts
- They are of relatively small size (approximately 12 kb) in circular form
- Are rendered linear and of very large size, up to several megabases (Mb), when introduced as cloning vectors in yeast.
-They are amenable to large-scale plasmid amplification in E. coli and to creation of specific genetic changes within the exogenous DNA sequence
https://www.researchgate.net/publication/228002816_Yeast_Artificial_Chromosomes

Remake of Yeast plasmids & Artificial Chromosomes (G1) by Víctor Manuel Rodríguez García

1. Characteristics (from the presentation and other sources)

2. Example (diagrams and biobricks)

3. Application (one example where this vector is used in GE)

4. References from websites and reviewed sources

Characteristics

YIPlac204TKC-GFP-HDEL

2. Example: YEp181-CUP1-His-Smt3

YRps

1. Natural occurring plasmids (2µm), 6.3 Kbp

Characteristics Yeps

References

YIplac204-TC-OM45-FKBPx4

Application

YCps - Example

Example (diagram and biobricks)

Charateristics

Aplication

YEps - Team 2

YEplac181-TAF14

Characteristic

1. generated by the insertion of bacterial plasmid sequences and appropriate selection markers

1. bear two characteristic sequences: autonomously replicating sequences (ARS) and centromeric (CEN) sequences

Application:

Characteristics

Applications

Applications YRps

Copies in YCps

Application

Types of integrative plasmids--

Applications

YRps

Copies in YEps

Queta

https://www.sciencedirect.com/topics/neuroscience/integrative-plasmid

HaloTag-bActinCDS-bActin3'UTR-MS2V5

2. pYAC example

3. Applications

Characteristics

Application

Characteristics

Application of BACs

Example of integrative plasmid

References:

pBacPAK8

pSmartBAC-S

pSmartBAC-S(Plasmid #107268)

pLV-SI-112

pLV-SI-112(Plasmid #131127)

Example-Application

pBS-TarHom-Tol2-kanaR-cryaa-dsRed

3. Applications

Yeast Artificial Chromosomes (YACs) [online] Microbe notes Available at: https://microbenotes.com/yeast-artificial-chromosomes-yacs/ [Accessed 8 April 2021]

​Cromosoma artificial de levadura (YAC) [online] Microbe notes Available at: https://www.genome.gov/es/genetics-glossary/Cromosoma-artificial-de-levadura [Accessed 8 April 2021]

pDY-HA1-HA2-neo

BACs - Example

MTK678_002

Applications

Characteristics

Alejandro Velázquez Velázquez team 3

Alejandro Velázquez Velázquez team 3

applications

Applications

characteristics

pSmartBAC-S
(Plasmid #107268)

pLV-SI-112
(Plasmid #131127)

Cromosoma artificial de levadura (YAC) [online] Microbe notes Available at: https://www.genome.gov/es/genetics-glossary/Cromosoma-artificial-de-levadura [Accessed 8 April 2021]