Team:TU-Munich/Results/KillSwitch

From 2013.igem.org

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The Kill Switch mechanism is the most complex and ambitious aspect of our project on the protein level. The DNA constructs are very large and even exceed the Part Registry´s frame (Fig. 1). For details on its design and function see [https://2013.igem.org/Team:TU-Munich/Project/Killswitch here]. We were very excited to start experimenting with the killswitch moss. However, when we opened the redlight filter foil, we found none of the allegedly transgenic moss lines had survived. There is a variety of possible reasons for this outcome that need to be discussed in order to improve our approach.
The Kill Switch mechanism is the most complex and ambitious aspect of our project on the protein level. The DNA constructs are very large and even exceed the Part Registry´s frame (Fig. 1). For details on its design and function see [https://2013.igem.org/Team:TU-Munich/Project/Killswitch here]. We were very excited to start experimenting with the killswitch moss. However, when we opened the redlight filter foil, we found none of the allegedly transgenic moss lines had survived. There is a variety of possible reasons for this outcome that need to be discussed in order to improve our approach.
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[[File:TUM13_PartsRegistryKillSwitch.png|thumb|center|750px| '''Figure 1''': [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159118 Registry entry] of the PhyB/PIF6 version of the Kill Switch, BBa_K1159118]]
==Transformation of the large DNA constructs==
==Transformation of the large DNA constructs==
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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''.
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''.
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[[File:TUM13_PartsRegistryKillSwitch.png|thumb|center|750px| '''Figure 1''': [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159118 Registry entry] of the PhyB/PIF6 version of the Kill Switch, BBa_K1159118]]
 
==Level of promoter activity==
==Level of promoter activity==
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[[File:PBI_376_f1.gif|thumb|right|500px| '''Figure 2''': 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]]]]
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[[File:PBI_376_f1.gif|thumb|right|400px| '''Figure 2''': 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]]]]
==Photosensitivity==
==Photosensitivity==

Revision as of 23:04, 28 October 2013


Kill Switch

The Kill Switch mechanism is the most complex and ambitious aspect of our project on the protein level. The DNA constructs are very large and even exceed the Part Registry´s frame (Fig. 1). 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 none of the allegedly transgenic moss lines had survived. There is a variety of possible reasons for this outcome that need to be discussed in order to improve our approach.

Figure 1: [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159118 Registry entry] of the PhyB/PIF6 version of the Kill Switch, BBa_K1159118

Transformation of the large DNA constructs

For the PEG-mediated moss transformation, we used plasmids linearized with EcoRI. Contrary to the targeted gene transfer described by [http://www.plant-biotech.net/paper/CurrGenet_2003_hohe.pdf Hohe et al., 2003], our constructs contain 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.

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 2: 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

Photosensitivity

Figure 3: Working with the photosensitive protoplasts


Figure 3: Working with the photosensitive protoplasts
Figure 3: Working with the photosensitive protoplasts
Figure 3: 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

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