Difference between revisions of "Part:BBa K1185002"
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     <td><img src="https://static.igem.org/mediawiki/2013/0/05/BareCillus_RFP_0mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/0/05/BareCillus_RFP_0mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/a/af/BareCillus_RFP_0mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 
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     <td><img src="https://static.igem.org/mediawiki/2013/1/11/BareCillus_RFP_0.05mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
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     <td><img src="https://static.igem.org/mediawiki/2013/c/c4/BareCillus_RFP_0.1mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/3/3e/BareCillus_RFP_0.1mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/3/3e/BareCillus_RFP_0.1mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
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     <td><img src="https://static.igem.org/mediawiki/2013/2/28/BareCillus_RFP_0.2mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/2/28/BareCillus_RFP_0.2mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/b/b3/BareCillus_RFP_0.2mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/b/b3/BareCillus_RFP_0.2mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
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     <td><img src="https://static.igem.org/mediawiki/2013/9/9b/BareCillus_RFP_0.4mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
     <td><img src="https://static.igem.org/mediawiki/2013/9/9b/BareCillus_RFP_0.4mM_IPTG_10mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">10 minutes</div></td>
 
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Revision as of 12:48, 25 September 2013

HBsu-RFP

This BioBrick codes for a HBsu protein attached to a red fluorescent protein (RFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the RFP through ten amino acid flexible linker sequence. This allows the observation of Bacillus subtilis DNA using fluorescence microscopy. The integration strategy that we opted for was to clone in the BioBrick into the Multiple cloning site of pMutin4 backbone. First we attached HindIII restriction sites on 5'end and SacII restriction sites on 3'end of the BioBrick. We then cut the pMutin4 backbone and BioBrick using the previously mentioned restriction enzymes, this allowed us to ligate the plasmid and BioBrick together. We also attached a ~300bp amyE homology region onto the 3'end of the BioBrick after the SacII to allow the single cross over and integration of the whole plasmid into the genome. The pMutin4 plasmid contains a ery+ resistance marker for B.subtilis and amp+ for E.coli and also contains lacI, lacZ and Pspac promoter which is an IPTG induced promoter which regulated the transcription of this BioBrick. An alternative method to use this part would be to clone this BioBrick out and use any Assembly protocol to attach a desired promoter, RBS and antibiotic resistance genes.

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 AgeI site found at 881
    Illegal AgeI site found at 993
  • 1000
    COMPATIBLE WITH RFC[1000]


Testing and Characterisation

To test that HBsu-RFP (BBa_K1185002) BioBrick works as designed; we transformed Bacillus subtilis str. 168 with the pMUTIN4 plasmid containing our HBsu-RFP construct.There were colonies on Plate 1, a LB + 5ug/ml ery plate containing the transformed B. subtilis with the plasmid containing our constructs as well as plate 2, which acts as our positive control. There are no colonies on the negative control plate (Plate 3) suggesting that the colonies on both Plate 1 and Plate 2 are indeed bacteria that has been successfully transformed with the plasmids instead of backgrounds. The results of the experiments are displayed in Figure 1.

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Plate 1: B. subtilis transformed with HBsu-RFP plated on LB + ery (5ug/ml) plate.
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Plate 2: B. subtilis transformed with pGFPrrnB plated on LB + ery (5ug/ml) plate(positive control).
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Plate 3: B. subtilis transformed with water plated on LB plate (viability of cells).
Figure 1. Transformation plates of ''Bacillus subtilis'' with HBsu-RFP, pGFPrrnB (as possitive control) and water as control to check the viability of cells.

In order to make sure that the BioBrick had been integrated properly into the endogenous amyE region, the transformants were plated out onto Starch plate. As the BioBrick integrated into the amyE region the cells did not produce amylase thus were unable to digest starch, and when iodine was applied to the cells they did not have a zone of clearance. As can be seen in Figure 2, both B. subtilis with either HBsu-sfGFP or HBsu-RFP constructs don't have any zone of clearance around the colonies in comparison to wild type B. subtilis str.168 and B. subtilis BSB1. The results of this iodine test further confirm that the transformants seen on Plate 1 Figure 1 are indeed colonies with our contruct integrated into the amyE region.


Figure 2. Starch plate showing alpha amylase activity. The ''B. subtilis'' 168 HBsu-GFP and HBsu-RFP colonies have no zones of clearance indicating that our HBsu-sfGFP and HBsu-RFP have integrated into the B. subtilis chromosome via the ''amyE'' region stopping starch digestion.

To gain evidence that our BioBrick is actually binding to DNA we stained the genome of B. subtilis cells transformed with our BioBrick with DAPI. The binds DNA and emits a blue fluorescence at 465nm. As can be seen in figure 3, the localisation of the DAPI coinsides with the red fluorescence emitted by HBsu-RFP at 610nm, suggesting that the HBsu-RFP is binding to the B. subtilis chromosome.


Figure 3. Left hand image shows B. subtilis Phase contrast image, the central shows the bacteria expressing HBsu-RFP and the right corner shows fluoresecense with DAPI staining. The HBsu-RFP and DAPI fluorescence shows same localisation in signal, indicating our BioBrick actually binds to DNA.

  • Figure 3

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    Figure 3. Negative control: Fluorescence microscopy showing how RFP expession changes with time on addition of 0mM IPTG in B.subtilis containing our HBsu-RFP BioBrick.

  • Figure 4

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    Figure 4. Fluorescence microscopy showing how RFP expession changes with time on addition of 0.05mM IPTG in B.subtilis containing our HBsu-RFP BioBrick.

  • Figure 5

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    Figure 5. Fluorescence microscopy showing how RFP expession changes with time on addition of 0.1mM IPTG in B.subtilis containing our HBsu-RFP BioBrick.

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    Figure 6. Fluorescence microscopy showing how RFP expession changes with time on addition of 0.2mM IPTG in B.subtilis containing our HBsu-RFP BioBrick.

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    Figure 7. Fluorescence microscopy showing how RFP expession changes with time on addition of 0.4mM IPTG in B.subtilis containing our HBsu-RFP BioBrick.

  • Figure 8

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    Figure 8. Fluorescence microscopy showing how RFP expession changes with time on addition of 0.8mM IPTG in B.subtilis containing our HBsu-RFP BioBrick.

References

[http://www.ncbi.nlm.nih.gov/pubmed/10224127 Kouji, N., Shou-ichi, Y., Takao, Y. & Kunio , Y., 1999. Bacillus subtilis Histone-like Protein, HBsu, Is an Integral Component of a SRP-like Particle That Can Bind the Alu Domain of Small Cytoplasmic RNA. The Journal of Biological Chemistry , Volume 274, pp. 13569-13576.]

[http://www.ncbi.nlm.nih.gov/pubmed/1902464 Micka, B., Groch, N., Heinemann, U. & Marahiel , M., 1991. Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA binding protein HBsu. Journal of Bacteriology, May;173(10), pp. 3191 -3198.]

[http://www.ncbi.nlm.nih.gov/pubmed/1382620 Micka, B. & Marahiel, M., 1992. The DNA-binding protein HBsu is essential for normal growth and development in Bacillus subtilis. Biochimie, 74(7-8), pp. 641-650.]

[http://www.ncbi.nlm.nih.gov/pubmed/10715001 Ross , M. & Setlow, P., 2000. The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA.. Journal of Bacteriology, 182(7), pp. 1942-1948.]