Team:TU-Munich/Results/KillSwitch

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Revision as of 00:07, 29 October 2013

Kill Switch

Figure 1: Dead Kill Switch moss protonema

The Kill Switch mechanism is the most complex and ambitious aspect of our project on the protein level. For details on its design and function see here. We were very excited to start experimenting with the killswitch moss.

However, when we opened the redlight filter foil, we found both the PIF3 and the PIF6 version of the allegedly transgenic moss lines were dead. There is a variety of possible reasons for this outcome that need to be discussed in order to improve our approach and produce live Kill Switch moss lines.

Transformation of the large DNA constructs

The Kill Switch DNA constructs are very large and even exceed the Part Registry´s frame (see Figure 2). For the PEG-mediated moss transformation, we used plasmids linearized with EcoRI, but contrary to the targeted gene transfer described by [http://www.plant-biotech.net/paper/CurrGenet_2003_hohe.pdf Hohe et al., 2003], our constructs contained no flanking homologuos regions. Our random integration transformation worked successfully for our other contructs, whereas the large Kill Switch construct might need further inquiry for a suitable integration site. Due to its size, the construct might be less stable and require sensitive handling. These factors could have lead to a failed transformation, in which case the moss would have died through the G418 selection process.

Figure 2: [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159118 Registry entry] of the PhyB/PIF6 version of the Kill Switch, BBa_K1159118

We weren´t yet able to express the Kill Switch in E.coli, which is most likely due to codon usage discrepancy. While Physcomitrella patens is not very dicriminating regarding codon usage, the function and expression of proteins designed for Physco is not guaranteed to work in E.coli.

Level of promoter activity

File:PBI 376 f1.gif

Figure 4: Comparative expression performance of different constitutive mammalian and plant promoters in Physcomitrella patens, http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009

We used the Actin 5 promotor from Physcomitrella patens [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306], which is the strongest known Physco promotor (see Figure 4). The very high intensity of expression might lead to problems concerning the vitality of the transgenic moss. Because the Kill Switch encodes for the very effective thermonuclease, overexpression might lead to loss of vitality in the moss regardless of its fixation to the cell membrane. Equipping the Kill Switch with a promotor for a lower level of expression, such as the CMV promotor or the SV40 promotor, could maybe lead to a higher survival rate of the transgenic moss.

Photosensitivity

Figure 6: Working with the photosensitive protoplasts

Figure 5: Working with the photosensitive protoplasts

References

http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009 Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries, Plant Biotechnol J. 2009 Feb;7(2):210

http://www.plant-biotech.net/paper/CurrGenet_2003_hohe.pdf Hohe et al., 2003 An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens, Curr Genet (2004) 44: 339–347

@@ Line 25: / Line 25: @@
 [[File:PBI_376_f1.gif|thumb|right|300px| '''Figure 4''': Comparative expression performance of different constitutive mammalian and plant promoters in Physcomitrella patens, [[http://www.ncbi.nlm.nih.gov/pubmed/19021876 Gitzinger et al., 2009]]]]
-We used the Actin 5 promotor from ''Physcomitrella patens'' [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306], which is the strongest known Physco promotor (see Figure 4). The very high intensity of expression might lead to problems concerning the vitality of the transgenic moss. Because the Kill Switch encodes for the very effective thermonuclease, overexpression might lead to loss of vitality in the moss regardless of its fixation to the cell membrane. Equipping the Kill Switch with a promotor for a lower level of expression could maybe lead to a higher survival rate of the transgenic moss.
+We used the Actin 5 promotor from ''Physcomitrella patens'' [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159306 BBa_K1159306], which is the strongest known Physco promotor (see Figure 4). The very high intensity of expression might lead to problems concerning the vitality of the transgenic moss. Because the Kill Switch encodes for the very effective thermonuclease, overexpression might lead to loss of vitality in the moss regardless of its fixation to the cell membrane. Equipping the Kill Switch with a promotor for a lower level of expression, such as the CMV promotor or the SV40 promotor, could maybe lead to a higher survival rate of the transgenic moss.
 ==Photosensitivity==