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Instructions: This cosmid collection
was prepared in the laboratory of Dr. Bruce Holloway at Monash University in
Australia and was later transferred to the laboratory of Dr. Paul Phibbs at
East Carolina University School of Medicine in Greenville, North Carolina.
The genomic DNA used was from the prototroph PAO1293 and the cosmid parent
was pLA2917. Cosmids are maintained in E. coli S17-1 frozen in rich
media with 15% DMSO (Dimethyl Sulfoxide) in microtitre trays at -70 degrees
Celsius. References: The cosmid library is
described in: Ratnaningsih, E., S. Dharmsthiti, V. Krishnapillai, A. Morgan,
M. Sinclair and B.W. Holloway, 1990. A combined physical and genetic map of
Pseudomonas aeruginosa PAO. Journal of General Microbiology 136:
2351-2357. Construction of the
cosmid parent pLA2917 is described in: Allen, L. and R.S. Hanson, 1985. Construction
of broad-host-range cosmid cloning vectors: identification of genes necessary
for growth of Methylobacterium organophilum on methanol. Journal of
Bacteriology 161: 955-962. E. coli S17-1 is described in: Simon,
R., V. Priefer and A. Puhler, 1983. A broad host range mobilisation system
for in vivo genetic engineering: transposon mutagenesis in gram negative
bacteria. Biotechnology 1:784-791. An excellent source for
listings of genetic markers, phenotypic traits affected, mapping data, and
references is: Holloway, B.W., U. Rohmling and B. Tummler, 1994. Genomic
mapping of Pseudomonas aeruginosa PAO. Microbiology 140:
2907-2929. There are four ways
to search the cosmid database: 1. Genetic Marker
Search: Similar to searching
the strain database by genotype, the more specific you are the less matches
you will see. For example, searching for "met" will yield more
matches than searching for "met-28". You will get a listing of all
cosmids known to complement mutations in this genetic marker and other
closely linked genetic markers. If you get no matches on your first attempt,
try using only the three letter marker designation. Only enter one marker
designation at a time in the search field. The database is searching for any
string of characters containing your search entry, and the chances of you
getting the marker designations, numbers, and spacing of more than one
genotype exactly the same as the database entry are very slight. 2. Map Location
Search: Map location numbers
need to be entered in a very specific way to successfully search the
database. All single digit designations must be converted to 2 digits by
adding a zero. (ie. minute 1 should be entered as 01) If you search for a
single digit, you will get results from all numbers that contain that digit.
(ie. a search for 2 will return results from minutes: 2, 12, 20, 32, etc.) 3. Cosmid Number
Search: Cosmid numbers need to
be entered in a very specific way to successfully search the database. All of
the cosmid designations must be complete to return results. Cosmids are
numbered using a simple system that corresponds to the method with which the
library was prepared. All designations begin with "pMO"
which stands for plasmidMOnash. This is followed by six digits.
The first two digits correspond to the strain from which the genomic DNA was
prepared. For this cosmid library strain PAO1293 was used, and the
designation for this strain is 01. The next two digits refer to the
microtitre tray in which each clone is located. There are forty trays,
numbered 01-40. The last two digits refer to the well in that tray,
numbered 01-50. Therefore the designation for the cosmid located in
tray 22, well 01 would be pMO012201. If you have found a
particular cosmid in the reference papers that you would like to order, you
will need to enter the complete designation to find it in the database. 4. SpeI Fragment
Search: SpeI fragment numbers
are found in data from: Ratnaningsih, et al, 1990 (complete reference above).
SpeI fragment numbers need to be entered in a very specific way to
successfully search the database. All single digit designations must be
converted to 2 digits by adding a zero. (ie. minute 1 should be entered as
01) If you search for a single digit, you will get results from all numbers
that contain that digit. (ie. a search for 2 will return results from
fragments : 2, 12, 22, etc.) How to use results: When you view the
matches to each of your searches, you will be given the opportunity to submit
requests for each of the cosmids. The last column of the report chart allows
you to check a box to add an available cosmid to your request. If you check boxes to
request available cosmids you will then proceed to the request form, by
clicking on the "Request Checked Cosmids" button, to mail your
request to the collection curator. At this point you can also reduce your
list of requested cosmids. Please fill out the request form completely and
submit it to have your request processed. After receiving your request, we
may write or call for more information. Please allow us several days to ship
your request to you. If you are working at a non-academic, for-profit
facility, a small fee of $25.00 per cosmid is required to cover our operating
and shipping expenses. An invoice will be sent in your shipment for you to
submit to your purchasing or Accounts Payable Department. If you have difficulty
searching the database or submitting your request, please feel free to e-mail
your request to the curator through the comments page,
or by writing to Pseudomonas@mail.ecu.edu.
You can also mail or fax requests or comments to:
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