Echinobase/bac information

Genomic BAC Resources

Libraries of genomic sequence fragments are maintained in bacterial artificial chromosome (BAC) vectors for seven lower deuterostome species (BAC Table). These libraries were originally constructed as part of the Sea Urchin Genome Project. On average, the insert sizes for our BAC libraries are ~140 kb. Thus, for the 800 Mb S. purpuratus genome, 1X coverage is encompassed by, on average, 5,700 clones. Each library consists of >100,000 clones, providing ~17X genome coverage.

A subset of the S. purpuratus BACs have been mapped to the v3.1 genome assembly. As part of the SUGP, about 8,000 BAC clones were end sequenced using Sanger technology. Genomic positions for these BACs are indicated as a track in JBrowse on the v5.0 genome assembly. An additional set of individual BACs were sequenced completely; links to the NCBI page are provided in the BAC Table. Finally, some BACs have been identified using hybridization screening of arrayed library filters (see BAC Table).

BAC Vector Sequence. The BACs are maintained in the pBACe3.6 vector which originates from Children's Hospital Oakland Research Institute in Oakland, California, USA. This vector confers resistance to the antibiotic chloramphenicol and is fully described by Frengen and colleagues. The vector sequence is available here.

Relevant publications: A sea urchin genome project: Sequence scan, virtual map, and additional resources. Cameron, RA, et al., 2000. Proc. Natl. Acad. Sci. USA. Bacterial artificial chromosomes as recombinant reporter constructs to investigate gene expression and regulation in echinoderms. Buckley, KM et al., 2017. Briefings in Functional Genomics.

BAC Protocols

Screening a BAC library

Generating a recombinant BAC

BAC miniprep protocol

Analyzing BAC DNA by PFGE

Recombinant BAC Resources Echinobase has generated and maintains a set of reporter BAC constructs in which a portion of the gene of interest is replaced by the sequence encoding a fluorescent protein (i.e. GFP, mCherry). Existing clones are available by request. We will also consider requests to make additional recombinant reporter constructs as time permits.

To request a recombinant or wildtype BAC for non-commercial use:

1. Identify the BAC of interest computationally or by screening our libraries.

2. Request the BAC by emailing Veronica Hinman.

BAC numbers eg. Sp_117A03_L

Sp = Species Sp, S. purpuratus

117 = Plate number

A = Plate row

03 = Plate column

L = long or S, short

Storing BACs

Short term

To avoid shearing the long BAC DNA fragments, isolated BACs should never be vortexed or frozen. Store BAC DNA at 4°C in TE (10 mM Tris, pH = 7.4; 10 mM EDTA).

Long term storage

Store glycerol bacteria stocks (15-25% glycerol) at -80°C.

Working with BACs

Antibiotic conditions for working with BAC clones

Kanamycin Stock (25 μg/μL): Dissolve 0.5 g of kanamycin into 10 ml of ddH2O.

Filter through a 0.22 µm filter to sterilize.

Aliquot and store at -20°C.

Use at 1:1000 dilution (25 μg/mL).

Chloramphenicol Stock (25 μg/μl): Dissolve in ETOH store at -20°C.

Use at 1:2000 dilution (12.5 μg/ml).

Transport to Pittsburgh

The BAC libraries were prepared using the Qbot.

The libraries fit into six -80°C freezers.

The truck had power outlets to plug the freezers for the cross-country drive.

The truck was loaded.

Ready to go!

The CIT Team.