Part:BBa_K914018

P1003** Kan resistant gene with 2 Amber Codon

The amber mutations avoid the expression of the P1003 gene even at usual concentration that prevent the expression of kanamycin resistance, although one mutation (BBa_K914009) permits some leakage in translation for this gene. This mutation is rescued with the tRNA amber suppressor supD (BBa_K914000). The idea here is to prevent the kanamycin gene resistance to be expressed in case horizontal gene transfer.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The positive and negative controls behave correctly, and similarly to the previous experiment (see K914009), it is also the case for the complementation of Kan** with supD. That means that two mutation are not a problem for the cell. Moreover, we notice that there is no more leakiness in the system at usual concentration, even at low concentration. Actually the double kanamycin resistant gene with two mutation behave exactly like the negative control (Plac + supD) which means that semantic containment is working well.

Figure 4A and 4B: The bar graph represent the OD₆₀₀ at time = 8h20' (black line Figure 4C) for the different strains at a given kanamycin concentration (black line on Figure 4B). On B, we observe the variations of the OD₆₀₀ at different kanamycin concentrations, at the same time.

Figure 4C: The variations of the OD₆₀₀ is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL).

Comparison of K914009 (Kan*) and K914018 (Kan**)

Here is a graph that gather both results, to highlight the effect of the second mutation. The results are normalized intra group (experiment with the one-mutated gene, and with the two-mutated gene) in order to have the same scale to compare them. We took the average of the positive (KanR+supD) and the negative (Plac+supD) controls.

Figure: Variation of OD₆₀₀ in different concentrations of [Kan] (µg/mL) at t= 8,37h

This part has been improved by part BBa_K2725012 - P1003 with 5 amber STOP codon substitutions. This addition of these extra amber STOP codons decreases the probability of read-through (and therefore expression) from 1 in every 10^4 translation attempts (P1003**) to 1 in every 10^10 translation attempts (P1003 5*). When expressed in combination with BBa_K2725016, P1003 5* can be expressed at a level that allows high level kanamycin resistance.