Part:BBa_K896012

Inv+LLO+RFP ( reporter for invasin& listeriolysin)

We improve the part from Group: iGEM10_Warsaw (part : <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K299812">BBa K299811</a>)

Ande here is what we had accomplished this year

Function of inv-llo gene on the platform of cyanobacteria

Author: Chiu Hsun-I from NYMU.Taipei, 2012

 Introduction

References revealed when engineered with invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes[1], new S. elongatus was able to invade into cultured mammalian cells and was capable of symbiosis with eukaryotic cells. The symbiosis possibility of induced pluripotent stem cells from mice and J774 mouse macrophage cell line were evaluated.

 Method and materials

Plasmid preparation

Invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes were provided by Pamela A. Silver, Harvard Medical school[1].

Symbiosis experiment

Three types of mammalian cells were evaluated—J774 mouse macrophage cell line and mouse induced pluripotent stem cells in embryonic stem(ES) cells stage and embryoid body(EB) stage.Synechococcus elongatus PCC 7942 was the first cyanobacterial strain which could be transformed by exogenously added DNA.Engineered cyanobacteria which were transformed inv-llo plasmid cocultured with cells under Leibovitz's L-15 medium without antibiotics. After 24 hours, supernatants were discarded and cells were washed with PBS two times and the medium was replaced by L-15 containing antibiotics. Cells were fixed by 4% paraformaldehyde and were observed by fluorescent microscope or confocal laser microscope after 24 hours. Detailed methods were described on NYMU.Taipei wiki.

 Discussion and Results

Under the fluorescence microscope 530-550 nm excitation filter, red pots were S. elongates PCC7942. Under the transmitted light, round and transparent one was cell.

Figure 1 revealed wildtype and inv-llo transformed type both entered into J774 macrophage cell line by phagocytosis and the help of inv-llo.

inv-llo strain, merged image of fluorescence and bright field<p>

wildtype, merged image of fluorescence and bright field

Figure 2 showed the coculture results of iPS cells at ES stage. Results showed that inv-llo transformation would increase the recruitment of cyanobacteria on induced pluripotent stem cells instead of the entry.

inv-llo strain, merged image of fluorescence and bright field.

wildtype, merged image of fluorescence and bright field.

On figure 3, there were similar and relative results of the iPS cells at EB stage to iPS cells at ES stage. The symbiosis of stem cells would be more difficult but the recruitment provided a potential for invading mammalian cells.

inv-llo strain, merged image of fluorescence and bright field

 Conclusion

The symbiosis possibility of induced pluripotent stem cells from mice and J774 mouse macrophage cell line were evaluated. Results showed that invasin and listeriolysin O transformation would raise the entry speed and escape rate in J774 mouse macrophage cell line, and would increase the recruitment of cyanobacteria on induced pluripotent stem cells.

 References

[1]Towards a Synthetic Chloroplast, Christina M. Agapakis,1 Henrike Niederholtmeyer,1,¤ Ramil R. Noche,1 Tami D. Lieberman,1 Sean G. Megason,1 Jeffrey C. Way,2 and Pamela A. Silver1,2,PLoS One. 2011; 6(4): e18877.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3936
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 770
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1387
  
• 1000
  COMPATIBLE WITH RFC[1000]