1. TT and TR Collections
Two separate collections exist: TT-Strains, whose numbers start with "TT", contain a transposon or are derived from a strain which contains a transposon (Tn10, Tn5, Mu derivatives, etc.). TR Strains, whose numbers start with "TR", have never been deliberately exposed to a transposable element.
2. Storage places
Depending on the stability of each particular strain, it is either stored in the frozen section or in stabs. The frozen section contains duplicates of each strain, stored side by side in freezer boxes. A key to the location of theses strains is kept in the notebook labeled "frozen strains". The stab part of the collection contains three copies of each strain, one for general use and two backups. One of the backups is locked.
3. What should be stored in the lab collection?
Strains should be put in the collection only if it is clear that they are correct and will probably be used in a publication. Avoid putting many independent isolates of the same strain or uncharacterized mutations in the lab collection. Keep individual collections for these strains.
B. Procedure for storing and cataloging a strain
1. Decide whether a strain should be in the TT or TR collection.
2. Determine whether a strain should be frozen or stored in a stab.
Any strain containing a Tn5, Tn10 or Mud should be frozen. Any unstable and most F' containing strains should be frozen. Standard auxotrophs can be stored in stabs.
3. Enter the strain in the appropriate strain book using the following guidelines.
a. Enter the parents of the strain (paternal and maternal).
b. Enter the complete genotype using standard nomenclature for alleles including the allele number and the locus letter (if appropriate). DO NOT write genotypes by referring to other strains or by describing phenotypes. Instructions for assigning allele numbers are discussed in section C.
c. Allele numbers assigned to new mutations must come from the large notebook labeled "stock book". This book has the allele numbers which have been assigned to this lab, along with other miscellaneous information. If the locus you need is absent or is out of numbers, call Ken Sanderson (403-220-6792) to obtain a new block of allele numbers. Be very sure to mark off the allele numbers that you use in the stock book.
d. Clearly mark E. coil strains in the left column and enter their genotypes in capital letter.
e. Enter the strain's original number from a personal strain collection so that mistakes in freezing or recording may be remedied.
f. DO NOT WRITE STRAINS IN THE BOOK UNLESS YOU HAVE STORED THEM IN THE COLLECTION!
4. Strains that are to be stored in the stab collection.
Make 3 labeled and wax-sealed copies of each strain. For those that are to be frozen, enter their TT or TR number in the frozen strains book (duplicates in adjacent columns), grow an overnight culture, put approximately 1.5ml of the fresh culture into each of two DMSO vial, clean the vials' caps to remove oil from the surface, then label with freezer label, and place them in the appropriate position of the correct freezer box.
C. Standard nomenclature
1. Mutational alleles
Enter allele followed by locus letter (if appropriate) followed by the allele number (e.g. hisD201). For alleles lacking a locus letter, insert a dash in place of the letter (e.g. his-203).
a. mutational insertions:
If the insertion is located within a gene, enter the allele, the locus letter, then the allele number, followed by two colons and the insertion element (e.g. hisH9601::MudA).
b. z-- numbers
Many insertions have been isolated that are not known to be mutations of any particular gene. All
such insertions are designated by a three-letter symbol starting with z; the second and third letters
designated approximate map position in minutes. The second position designates 10-minutes map
segments numbered clockwise from minute 0 (a=0-10; b=10-20; c=20-30, etc.); the third letter
similarly designated minutes within any 10 minute segment. For example, an insertion located
between minutes 2 and 3 would be designated by the symbol "zac".
Allele numbers are assigned serially to such insertions regardless of the letters appearing in positions 2 and 3. Thus, if more refined mapping data warrants a new three-letter symbol, the numerical identity of the insertion is not lost. As with other alleles, "z" numbers are assigned from the stock book.
For proper nomenclature, the three letter symbol is followed by a dash, the "z" allele number, a
double colon, then the insertion element. For example, a Tn10 insertion located between minutes 2
and 3 would be designated zac-1::Tn10. Insertions on extra-chromosomal elements are designated
with "zz" followed by letter denoting the element used. For example, zzf denotes insertions on a
c. currently accepted designations for insertion elements.
Our lab's established designations for common insertion elements are listed below. Published
designations may differ from one journal to another.
Tn10d-tet; Tn10d-can; Tn10d-kan
MudA; MudF; MudJ; MudP; MudQ
Since chromosomal rearrangements involve more than one gene or gene cluster, they are distinguished from standard mutational alleles by a three capital letter symbol, CRR, in they are given a three letter symbol, CRR. A CRR symbol is assigned with an allele number regardless of the type of rearrangement. Upon characterization, it may be redesignated as a inversion (INV), a duplication (DUP), a deletion (DEL) or a transposition (TNP). It remains the same allele number to prevent confusion. The allele number is followed by a detailed description of the genotype in brackets.
Deletions are designated either by standard mutation nomenclature (above) or be rearrangement nomenclature according to the following rules. Standard mutation nomenclature may be used to describe deletions which remove one or more genes within an operon. Rearrangement nomenclature is appropriate when two or more unrelated genes are involved or for deletions generated by a transposable element and the gene.
The nomenclature for rearrangement formed from homologous recombination between Mud prophages for other transposons is illustrated by the following examples.
DEL443[(hisD9953)*Mud*(hisF9954)] is a deletion caused by recombination between the MudA insertions in hisD and hisF. This deletion would have its end points at the site of the original Mud insertions (hisD and hisF) and would have a MudA at the join point.
The same nomenclature is used for duplications. DUP442[(hisD9953)*MudA*(hisF9954) is a duplication of the material between the Mud insertions in hisD and hisF where a MudA exists at the join point.
4. Nomenclature references
Demerec M.; Adelber, E,; Clark, A.J.; Hartman, P.E. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54:61-76, 1966.
Hughes, K.; Roth, J. Directed formation of deletions and duplications using Mud(Ap, lac). Genetics 109:263-282, 1985.
Schmidt, M. Bacterial rearrangement nomenclature. Thesis, Appendix II: 163-175.
D. Using stab cultures
Racks of stabs should not be removed from the library. Remove only the vial(s) that you need and return them as soon as possible. Each time you use a stab, reseal it with hot wax and mark the label. If the label has four marks, make a new stab. Put the strain racks back where they belong, not the most convenient place for you!
E. Strain books
The genotypes of strains are kept in notebooks and in a computer file for easy reference. The strain books should not be removed from the library! These books will always remain the ultimate reference containing special notes about the strains and their parents. It is critical that they be taken care of with a reasonable amount of compulsiveness.