Part:BBa_K4219000:Design
Evolutionary-stabilized optimized GFP reporter protein
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 1004 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal NheI site found at 29
Illegal NheI site found at 52
Illegal SpeI site found at 1005
Illegal PstI site found at 1019
Illegal NotI site found at 7
Illegal NotI site found at 1012 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 1005 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal XbaI site found at 16
Illegal SpeI site found at 1005
Illegal PstI site found at 1019 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The sequence of this part was determined by utilizing the ESO software, developed by Staubility, TAU representative group for the iGEM 2020 competition. In order to validate that the optimization process has worked properly, an evolutionary experiment, checking whether a significant difference in the evolutionary stability between the original non-optimized part and the newly-optimized one might be witnessed, had been designed. Throughout which, we have measured the fluorescence levels of both parts (encoding for the GFP protein) and checked whether the loss of fluorescence (indicating loss of functionality and a decreased evolutionary stability) of the original part was significantly faster than that of the optimized one.
A full description of the part:
Selected Part Name: BBa_K4219000. Part Type: Coding. Short Description: Evolutionary-stabilized optimized GFP reporter protein. Long Description: This part is an optimized version of the BBa_K079050 construct. Our team optimized the old part and constructed the new one while utilizing the ESO software (Menuhin et al, 2022). The ESO is a software constructed by TAU IGEM team 2020 as part of their project, and we used this software with their help and under their guidance. The software optimizes the evolutionary stability of a given DNA construct, by preventing sites that are prone to mutation and recombination while preserving the amino acid sequence. While designing this new part, the goal stood in front of our eyes was to induce minimal changes, so the functionality of the initial construct will be preserved, while the evolutionary stability of the construct greatly elevated. This was verified by a lab evolution experiment, (please refer to the “improvement of an existing part” page for further details). The users of that part should address it exactly the same as they were addressing the original one – use it as a GFP protein constituting a DNA damage reporter – while taking into account that it is evolutionary-stabilized, namely will preserve its functionality for a longer period of time. The Source of the Part: BBa_K079050 that our team received from the iGEM 2021 Distribution kit. Design Considerations: The sequence of this part was determined by utilizing the ESO software, developed by 2020 TAU IGEM team. The software changes the DNA sequence in order to prevent sites that are prone to mutation or recombination while preserving the amino acid sequence. Our team created two variations of each part, one that only optimized the evolutionary stability of the construct, and a second one that also optimized the sequence for E.coli expression (codon usage optimization; for further information please refer to the “improvement of an existing part” page). Our team manually checked the work of the algorithm by MSA, to verify that indeed the amino acid sequence was preserved. Length: 1024 base pairs.
Source
The source of this part is, as already stated, the BBa_K079050 construct, stored in the 23G position, plate 3 of the iGEM 2021 Kit Distribution.