Part:BBa_K2118004
J23119 + FMS1 gene + PatA gene
It is a device is composed by J23119-FMS1-PatA parts. This genetic machine is the combination of the regular consitutive promoter and two enzymes that degrade the 3 basic types of polyamines; the Polyamine Oxidase (spermine and spermidine) and the Putrescine Aminotrasnferase (putrescine). The combination of three is a powerful polyamine degrading engine, which we have successufully clonned and transformed in E. coli and that we are submitting to the part registry as the full composite, but also in separated parts. The two genes incorporate the standard medium RBS BBa_B0032 upstream to the actual codifying sequence. Sanger sequencing provided by our sponsor GATC biotech has confirmed the integrity of the samples.
To measure the effectiveness of the device, we have run a 96-well plate reading for 12 hours to compare the growth of polyamine auxotroph cells with and without our device in M9 medium with a gradient of polyamines. From the literature, we decided that the reliable range of polyamine concentration would be between 0mM to 2mM, since these molecules are harmful, even for bacteria, when in high amounts.
Several conclusions were extracted from the pictures:
- We can verify that, indeed, the polyamine auxotroph cells require polyamines for their growth, since the group without polyamines is not able to grow neither with or without the device. We can appreciate a certain growth during the first hours, probably because of the intracelular reservoirs of polyamines, that are exhausted soon.
- The cells with the device experience a significant overall reduction in growth rate and total optical density. This is certainly produced due to the expression of two constitutive genes, which is a metabolic burden for the cell that reduces its reproductive capacities.
- The device group with low to medium concentration of polyamines experiences a sudden delay in their growth between hours 4 and 5. This can be explained by the fact that they are polyamine auxotroph but still degrade their own vital compound. This leads to a critical point where there are not enough polyamines for the expanding population of cells and their growth is stopped.
- We can verify that an excess in polyamines (2mM) ends up being toxic for the cells in both cases. Moreover, the degradation of polyamines by the device group produces peroxide, which is toxic for cells when above 1μM and accelerates the death of the cells further.
- The above effect is less significant in the cases with polyamine concentrations 0.5 and 1μM, since the reservoir of these molecules in the medium is large enough for the cells to live even if they are degrading it.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1984
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 217
Illegal AgeI site found at 900
Illegal AgeI site found at 1162
Illegal AgeI site found at 1350
Illegal AgeI site found at 2300
Illegal AgeI site found at 2441
Illegal AgeI site found at 2456 - 1000COMPATIBLE WITH RFC[1000]
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