Difference between revisions of "Part:BBa K2136014"

 
 
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Resistance gene for algae expression. Bleomycin gives a resistance against zeocin, because ble binds to zeocin blocking its funtion.
 
Resistance gene for algae expression. Bleomycin gives a resistance against zeocin, because ble binds to zeocin blocking its funtion.
  
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<html>
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<p class="fig-label"> Figure 1: Gel electrophoresis of mCherry+pSB1C3 construct</p>
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<p>After ligating the sequence in the pJP22, we wanted to find the best way to transform C. reinhardtii, we tested Sapphire Blue, TAP medium and water as buffers during the electroporation. Then, cells were plated in Agar-TAP medium Petri dishes with different concentrations of Zeocin (an antibiotic from Bleomycin family) because the plasmid used had a sequence for Bleomycin resistance.</p>
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<img src="https://static.igem.org/mediawiki/2016/6/6c/T--USP_UNIFESP-Brazil--mCherry_placaA5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
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<img src="https://static.igem.org/mediawiki/2016/e/ef/T--USP_UNIFESP-Brazil--mCherry_placaA10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
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<br>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Figure 2: pJP22 mCherry transformants - Zeocin 5 ug/mL &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Figure 3: pJP22 mCherry transformants - Zeocin 10 ug/mL<br>
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<img src="https://static.igem.org/mediawiki/2016/d/d9/T--USP_UNIFESP-Brazil--mCherry_placaM5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
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<img src="https://static.igem.org/mediawiki/2016/7/77/T--USP_UNIFESP-Brazil--mCherry_placaM10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" /> <br>
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&nbsp; &nbsp; &nbsp; &nbsp; Figure 4: pJP22 mCherry transformants - Z5 (Water Transformed)&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Figure 5: pJP22 mCherry transformants - Z10 (Water Transformed)
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<img src="https://static.igem.org/mediawiki/2016/3/35/T--USP_UNIFESP-Brazil--mCherry_placaS5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
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<img src="https://static.igem.org/mediawiki/2016/d/da/T--USP_UNIFESP-Brazil--mCherry_s10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" /> <br>
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&nbsp; &nbsp; &nbsp; &nbsp;Figure 6: pJP22 mCherry transformants - Z5 (TAP Transformed) &nbsp;  &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Figure 7: pJP22 mCherry transformants - Z10 (TAP Transformed)
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<img src="https://static.igem.org/mediawiki/2016/e/e7/T--USP_UNIFESP-Brazil--mCherry_placaT5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
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<img src="https://static.igem.org/mediawiki/2016/5/5a/T--USP_UNIFESP-Brazil--mCherry_placaT10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" /> <br>
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pJP22 Figure 8: mCherry transformants - Z5 (Sapphire Transformed) &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Figure 9: pJP22 mCherry transformants - Z10 (Sapphire Transformed)<br><br>
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<p>As expected, the number of colony-forming units is much higher in the media with less Zeomicin.</p>
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<p>At the same time, the transformation of cells were not so good using Sapphire Buffer in comparison with Water and TAP medium.</p></html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 05:03, 19 October 2016


Bleomycin resistance gene

Resistance gene for algae expression. Bleomycin gives a resistance against zeocin, because ble binds to zeocin blocking its funtion.

Figure 1: Gel electrophoresis of mCherry+pSB1C3 construct

After ligating the sequence in the pJP22, we wanted to find the best way to transform C. reinhardtii, we tested Sapphire Blue, TAP medium and water as buffers during the electroporation. Then, cells were plated in Agar-TAP medium Petri dishes with different concentrations of Zeocin (an antibiotic from Bleomycin family) because the plasmid used had a sequence for Bleomycin resistance.


           Figure 2: pJP22 mCherry transformants - Zeocin 5 ug/mL                    Figure 3: pJP22 mCherry transformants - Zeocin 10 ug/mL

        Figure 4: pJP22 mCherry transformants - Z5 (Water Transformed)                Figure 5: pJP22 mCherry transformants - Z10 (Water Transformed)
       Figure 6: pJP22 mCherry transformants - Z5 (TAP Transformed)               Figure 7: pJP22 mCherry transformants - Z10 (TAP Transformed)
pJP22 Figure 8: mCherry transformants - Z5 (Sapphire Transformed)                  Figure 9: pJP22 mCherry transformants - Z10 (Sapphire Transformed)

As expected, the number of colony-forming units is much higher in the media with less Zeomicin.

At the same time, the transformation of cells were not so good using Sapphire Buffer in comparison with Water and TAP medium.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 774
    Illegal NgoMIV site found at 835
  • 1000
    COMPATIBLE WITH RFC[1000]