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    Genetic Engineering Assignment Bacterial Artificial Chromosome (Bac) and Yeast Artificial Chromosome (Yac)

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    Hairil Fiqri
    BACs and YACs are both artificial chromosome vectors used in genetic engineering. BACs can hold inserts up to 300kb in bacteria, are easy to purify and transform, and are very stable with low copy numbers. Common BAC vectors include pBeloBAC11 and pBACe3.6. YACs can hold much larger inserts over 2Mb in yeast but are less stable, more prone to rearrangement, and more difficult to transform and purify than BACs. Both vector types have their advantages and limitations for cloning DNA fragments.

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    Genetic Engineering Assignment Bacterial Artificial Chromosome (Bac) and Yeast Artificial Chromosome (Yac)

    Uploaded by

    Hairil Fiqri
    55 views5 pages
    BACs and YACs are both artificial chromosome vectors used in genetic engineering. BACs can hold inserts up to 300kb in bacteria, are easy to purify and transform, and are very stable with low copy numbers. Common BAC vectors include pBeloBAC11 and pBACe3.6. YACs can hold much larger inserts over 2Mb in yeast but are less stable, more prone to rearrangement, and more difficult to transform and purify than BACs. Both vector types have their advantages and limitations for cloning DNA fragments.

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    BACs and YACs are both artificial chromosome vectors used in genetic engineering. BACs can hold inserts up to 300kb in bacteria, are easy to purify and transform, and are very stable with low copy numbers. Common BAC vectors include pBeloBAC11 and pBACe3.6. YACs can hold much larger inserts over 2Mb in yeast but are less stable, more prone to rearrangement, and more difficult to transform and purify than BACs. Both vector types have their advantages and limitations for cloning DNA fragments.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    55 views5 pages

    Genetic Engineering Assignment Bacterial Artificial Chromosome (Bac) and Yeast Artificial Chromosome (Yac)

    Uploaded by

    Hairil Fiqri
    BACs and YACs are both artificial chromosome vectors used in genetic engineering. BACs can hold inserts up to 300kb in bacteria, are easy to purify and transform, and are very stable with low copy numbers. Common BAC vectors include pBeloBAC11 and pBACe3.6. YACs can hold much larger inserts over 2Mb in yeast but are less stable, more prone to rearrangement, and more difficult to transform and purify than BACs. Both vector types have their advantages and limitations for cloning DNA fragments.

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    © All Rights Reserved
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    of 5

    GENETIC ENGINEERING ASSIGNMENT

    BACTERIAL ARTIFICIAL CHROMOSOME (BAC)


    AND YEAST ARTIFICIAL CHROMOSOME (YAC)

    Venisa Yosephi 155100500111010


    Hairil Fiqri 155100500111019

    MAYOR AGRICULTURAL PRODUCT


    FACULTY OF AGRICULTURAL TECHNOLOGY
    BRAWIJAYA UNIVERSITY
    MALANG
    2017
    Bacteria Artificial Chromosome

    BAC vectors are plasmids constructed with the replication origin of E. coli F factor.
    Bacterial artificial chromosomes (BACs) are optimal for harboring long fragments of genomic
    DNA or large cDNA up to 300 kb in size. BAC libraries have been exploited for the development
    of detailed genetic and physical maps of major crops and positional cloning of genes of interest.
    Because of their high stability, low chimerism, and easy DNA purification, large-insert BAC
    libraries, and especially plant-transformation competent binary BAC (BIBAC) libraries have
    emerged as the large-insert arrayed libraries of choice for plant genome research. BACs are
    available to produce transgenic cells and animals for the functional studies of genes. BAC has 2
    origin of replication (ORI) but has a low copy number.

    BACs are useful for the construction of genomic libraries but their range of use is vast.
    Application of BACs as tools span basic science, economically rewarding industrial research,
    and fields as prosaic as animal husbandry. These are some examples of BAC:

    a. pBeloBAC11The primary characteristic of pBeloBAC11 is the addition of a lacZ gene into


    the multiple cloning site 2 of pBAC108L. On plates supplemented with X-gal/IPTG, an
    intact lacZ gene encodes b-galactosidase which catalyses the supplemented substrate
    into a blue substance. Successful ligation of insert DNA into the vector inactivates lacZ;
    hence, white colonies indicate the presence of a successful vector-insert ligation. How-
    ever, pBeloBAC11 is still a low-copy number plasmid from the presence of parA and
    parB
    b. pBACe3.6 is also based on pBAC108L but is more highly modified than pBeloBAC11 6.
    In order to address the issue of low plasmid copy numbers, the P1 replicon in F was
    deleted and a removable high copy number replicon originating from an inserted pUC19
    was introduced. Additionally, a different positive selection mechanism from blue/white
    selection used in pBeloBAC11 is employed. pBACe3.6 includes a 2.7 kb pUClink stuffer
    fragment flanked by two sets of six restriction sites within a sacB region. The sacB gene
    product is levansucrase, which converts sucrose, supplemented in the media, to levan,
    which is toxic to E. coli host cells. Hence, if the vector is religated without an insert, the
    functional sacB produces levansucrase and the cells die before forming colonies.
    Successful ligation of an insert into the vector increases the distance from the promoter
    to the coding region of sacB, disrupting toxic gene expression in the presence of
    sucrose.

    Each BAC vector has its own features depending on what the vector used for. The table
    below shows the features, cloning sites, and recombinant selection of each BAC:
    Yeast Artificial Chromosome

    Yeast articial chromosomes (YACs) are plasmid shuttle vectors capable of replicating
    and being selected incommon bacterial hosts such as Escherichia coli, as well as in the budding
    yeast Saccharomyces cerevisiae. They are of relatively small size (approximately 12kb) and of
    circular form when they are amplied or manipulated in E. coli, but are rendered linear and of
    very large size, i.e. several hundreds of kilobases (kb), when introduced as cloning vectors in
    yeast. Their capacity to accept large DNA inserts enables them to reach the minimum size
    (150kb) required for chromosome-like stability and for delity of transmission in yeast
    cells.YACs have several advantages over other large capacity vectors: these include
    accommodation of DNA segments thousands of kilobases in size and stable maintenance of
    cloned eukaryotic DNA due to the compatibility with the yeast replication machinery. These are
    subsequently ligated with the appropriate DNA insert before transformation into recipient yeast
    cells converted to spheroplasts, where telomere sequences are added by the cells telomerase
    enzymes.

    The basic structural features of YACs were developed from the yeast centromere
    shuttle-plasmids YCp series. These are composed of double-stranded circular DNA sequences
    carrying the b-lactamase gene bla and the bacterial pMB1 origin of replication, thus conferring
    resistance to ampicillin and the ability to replicate in bacteria, respectively. They also include
    yeast ARS1 with its associated CEN4 DNA sequence, as well as the URA3 selectable marker.
    On this basic scaold plasmid the yeast HIS3 is cloned, anked by a telomere-like DNA
    sequence derived from the termini of the Tetrahymena pyriformis macronuclear ribosomal DNA,
    which allows the formation of functional telomeres in yeast, that are adjacent to two recognition
    sites for the BamHI restriction enzyme. Most of these YACs also contain the cloning site in the
    middle of the SUP4 suppressor of an ochre allele of a tyrosine transfer RNA (tRNA) gene; this
    enables restoration of the normal white colour phenotype in otherwise red ade1 and/or ade2
    nonsense mutants insertional inactivation cloning process, the SUP4 gene is disrupted by the
    DNA insert, thus removing the suppression of the ade mutations and allowing their phenotypic
    expression as red colour.
    BACs vs YACs

    Yeast artificial chromosomes (YAC) can accommodate insert sizes in excess of 2 megabases
    (Mb) which vastly overcome the size limitation of previous vectors. However, yeast spheroplast
    transformation is relatively inefficient, and large amounts of DNA are required for library
    construction 10. YAC DNA, in ad-dition, is linear and can be difficult to isolate intact due to its
    susceptibility to shear. Most importantly, YAC clones are often unstable and chimeric 11 in
    nature and sequences with repetitious elements are prone to rear-rangement 12 or are un-
    clonable.Bacterial artificial chromosomes overcome many of the problems involved with YACs
    1. BACs can be transfected into E. coli by electroporation at efficiencies up to 100 times greater
    than yeast transformation. BAC DNA exists in supercoiled circular form that permits easy
    isolation and manipulation with minimal break-ing. In addition, clones can be effortlessly isolated
    via miniprep alkaline lysis and directly re-introduced into bacterial cells. Importantly, bacterial
    recombination systems are well characterized and recombination deficient strains of E. coli are
    readily available. It is not surprising, then, that BAC DNA is very stable, a trait that is aided by
    the low copy numbers maintained in each cell. However, there are BAC vectors that can attain
    very high copy numbers while maintaining DNA stability 6. One drawback of BAC vectors
    compared to YAC vectors is that the maximum insert size that BACs can accommodate merely
    exceeds about 300kb although clones in the mid-300 kb range are obtainable. Additionally, the
    number of successfully generated clones decreases when attempting to achieve higher insert
    sizes (personal observation) and there has been suggestion that there are species-specific
    library insert-size limitations based on base-pair content and sequence dissimilarities.

    Despite YACs having the ability to clone sizes up to 1MB of insert many problems have been
    detected including: chimerism, tedious steps in library construction and low yields of Y AC insert
    DNA. Thus a new E.coli based system have been developed, the BAC system (Yu et al., 2000),
    which offers many potential advantages over Y ACs.

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