Part:BBa_K4604041
piG_11 (tetR_kanR_riboK12_mazF)
Construct piG_11 is made up of the tet-promoter (consisting of the tet repressor and tet operator), that can be induced with doxycycline, the modified T7 RBS, a kanamycine resistance, a riboswitch for AdoCbl, mazF (toxin) and the rrnB terminator. The backbone we used in our experiments was pGGAselect. The kanamycine resistance was taken from iGEM UCDavis 2021 (BBa_K4094020), the tet operator from iGEM Freiburg 2022 (BBa_K4229059). Working with this BioBrick was discontinued after realizing the illegal restriction site in the kanR, resulting in a non-uploadable part.
Our vision with this BioBrick
The most susceptible sites for mutations in our CELLECT plasmid (BBa_K4604023) are the toxin gene mazF and the TetR promoter in front of bluB and mazF. The cells are exposed to a selection pressure, favoring random mutations that stop production of the toxin, new subpopulations with mutations of these plasmid parts could emerge. With sequencing results repeated over a time span of around four days, we were able to show that the toxin gene did not accumulated mutations which could have affected the function of the toxin. Even if the concerns of toxin mutation do not seem to have any impact on our system in the period we have tested, we are interested in finding a solution to minimize the probability of mutations that could affect our toxin long term. Implementing CELLECT (our system) in a bioreactor would require solid stability for long periods of time and numerous cell generations. Therefore, we would introduce a second selection marker. As an example, placing a kanamycin resistance gene in front of the riboswitch followed by the toxin under the same promoter. In theory, if the TetR promoter in front of the riboswitch and toxin gene mutated and therefore transcription of this part was stopped, the kanamycin resistance would also not be expressed, making a selection of cells possible. However, the promoter we used for testing this BioBrick appeared to be leaky. Therefore during initial experiments with the kanamycin resistance, we experienced that even without induction via doxycycline, cells survived in media supplemented with kanamycin. The solution would be to replace the leaky TetR promoter with a tighter inducible promoter. Optionally a higher kanamycin concentration could be used to counteract the leaky expression, as induction still increases expression of the resistance gene. Despite having these options to improve our system in mind, time was figuratively slipping through our fingers. Therefore we decided to focus on our system without the kanamycin resistance firstly, not testing this BioBrick further.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 913
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 913
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 913
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 913
Illegal NgoMIV site found at 1364
Illegal NgoMIV site found at 1700 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2335
Illegal SapI.rc site found at 1213
Illegal SapI.rc site found at 1423
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