Part:BBa_K3519009

medium promoter-traT

This part has the traT gene (BBa_K3519003) downstream to the medium strength Anderson promoter (BBa_J23118), the strong RBS (BBa_B0034) and is followed by the double terminator (BBa_B0015). Constitutive overexpression of the traT gene helps reduce the formation of stable mating pairs. The data provided below are from previous overexpression studies in the literature.

Usage and Biology

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Plasmid pRS31that expresses F sex factor DNA that includes traS, traT, traD, and traI was taken to study the surface exclusion property. F' cells are 100- to 300-fold worse recipients in conjugation than F-cells are to surface exclusion. pRS31 expresses surface exclusion more strongly than the F factor itself. Cells carrying pRS31 are found to be 10,000-fold worse recipients than F- cells are. Plasmids containing other regions of F DNA, such as pRS27 or pRS29, have no effects on recipient ability. The F' sex factor, JCFL0, is a lacI mutant of the Flac plasmid F42. A Study was carried out on of the amount of aggregation and DNA transfer found in matings between an Flac or Hfr donor and strains carrying pRS31 mutated in either traS or traT:

1. The traS mutants (pBE5 to pBE8) display a marked increase in recipient ability. DNA transfer into them is only 16-26 times worse than into cells carrying the control plasmid pRS27, whereas for the wild-type pRS31 it is 33,000 times worse (Table-1). There is, however, no corresponding increase in the percentage of mating aggregates. This remains at approximately 18%, the same level as in the wild type, and considerably below the 75% found in control matings. From these findings it can be said that the depressed level of aggregation in pRS31 and its traS mutants is because of the effect of the traT gene product.
2. The behavior of the traT mutants, pBE21 to pBE24, supports the above results as they all have increased levels of aggregation (Table-1). The DNA transfer into these traT cells was 90-215 times worse than the control even though the cells are in stable mating aggregates.

Table 1. Mating aggregation with surface exclusion-deficient mutants[1]





Fig. 1. NaDodSO4 gel electrophoresis of whole membrane preparations. Molecular weight standards are indicated by 39,000 (RNA polymerase a-subunit), 33,000 (purified protein II*), 21,000 (trypsin inhibitor) and 10,500 (purified F pili subunits). The following slot numbers correspond to cells carrying the plasmids indicated in parentheses: 3 (pBE44; traD14); 4 (pRS31); 5 (pBE5); 6 (pBE6); 7 (pBE7); 8 (pBE8); 9 (pBE21); 10 (pBE22); 11 (pBE23); 12 (pBE24). The corresponding tra mutations are indicated in Table 1. F+ indicates the Flac element JCFLO[1]

References:

1. Achtman, M., Kennedy, N., & Skurray, R. (1977). Cell--cell interactions in conjugating Escherichia coli: role of traT protein in surface exclusion. Proceedings of the National Academy of Sciences of the United States of America, 74(11), 5104–5108. https://doi.org/10.1073/pnas.74.11.5104

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 703
  Illegal BsaI.rc site found at 185