Difference between revisions of "Part:BBa K1185001"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1185001 short</partinfo>
 
<partinfo>BBa_K1185001 short</partinfo>
  
 
This BioBrick codes for a HBsu protein attached to a superfolded green fluorescent protein (sfGFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the sfGFP 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.
 
This BioBrick codes for a HBsu protein attached to a superfolded green fluorescent protein (sfGFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the sfGFP 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.
 +
 +
==HBsu-sfGFP Testing and Characterisation==
 +
 +
To test that HBsu-sfGFP [https://parts.igem.org/Part:BBa_K1185001 (BBa_K1185001)]BioBrick works as designed; we transformed ''Bacillus subtilis'' str. 168 with the  pMUTIN4 plasmid containing our HBsu-sfGFP construct. There were colonies on Plate 1 which is LB + 5ug/ml ery plates containing the transforms ''B. subtilis'' with the plasmid containing our constructs as well as plate 2 which act as our positive control. There is 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 display in Figure 1.
 +
<html>
 +
<style type="text/css">
 +
#body img{
 +
    margin-bottom:3px;
 +
}
 +
table{
 +
border:2px solid black;
 +
margin-left:50px;
 +
margin-bottom: 10px;
 +
width: 650px;
 +
}
 +
.italic{
 +
font-style: italic;
 +
}
 +
table.borderless {
 +
border:0px solid white;
 +
}
 +
 +
</style>
 +
<body>
 +
<br>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/5/5d/Barecillus_LB_ery_HBsu-GFP_1308.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 1: B. subtilis transformed with HBsu-sfGFP plated on LB + ery (5ug/ml) plate.</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/0/0f/Barecillus_LB_ery_168rrnB_1308.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 2: B. subtilis transformed with pGFPrrnB plated on LB + ery (5ug/ml) plate(positive control).</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/f/fb/Barecillus_LB_ery_168H20_1308.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 3: B. subtilis transformed with water plated on LB plate (viability of cells).</div></td>
 +
    <td><div class="italic">Figure 1. Transformation plates of ''Bacillus subtilis'' with HBsu-sfGFP, pGFPrrnB (as possitive control) and water as control to check the viability of cells.</div></td>
 +
  </tr>
 +
</table>
 +
</body>
 +
</html>
 +
 +
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 were indeed colonies with our contruct integrated into the ''amyE'' region.
 +
 +
<html>
 +
<table class = "borderless">
 +
<tr>
 +
<td align="center">
 +
<img src="https://static.igem.org/mediawiki/2013/e/ec/BareCillus_successxFPint.jpg" height="208px" width="268px">
 +
<br>
 +
<i>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.</i>
 +
</td>
 +
</tr>
 +
</table>
 +
</html>
 +
 +
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 green fluorescence emitted by HBsu-sfGFP at 560nm, suggesting that the HBsu-RFP is binding to the ''B. subtilis'' chromosome.
 +
 +
<html>
 +
<table class = "borderless">
 +
<tr>
 +
<td align="center">
 +
<img src="https://static.igem.org/mediawiki/2013/6/6b/BareCillus_New_DAPI_GFP_1_w3DAPI_465.jpg" height="250px" width="600px">
 +
<br>
 +
<i>Figure 3. Left hand image shows B. subtilis Phase contrast image, the central shows the bacteria expressing HBsu-sfGFP and the right corner shows fluoresecense with DAPI staining. The HBsu-sfGFP and DAPI fluorescence shows same localisation in signal, indicating our BioBrick actually binds to DNA.</i>
 +
</td>
 +
</tr>
 +
</table>
 +
</html>
 +
 +
<html>
 +
<style type="text/css">
 +
#body img{
 +
    margin-bottom:3px;
 +
}
 +
table{
 +
border:2px solid black;
 +
margin-left:50px;
 +
margin-bottom: 10px;
 +
width: 650px;
 +
}
 +
.italic{
 +
font-style: italic;
 +
}
 +
 +
</style>
 +
<body>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/7/78/BareCillus_GFP_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/2013/3/3b/BareCillus_GFP_0mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/a/a6/BareCillus_GFP_0mM_IPTG_60mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">60 minutes</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/f/f3/BareCillus_GFP_0mM_IPTG_120mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">120 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/c/c0/BareCillus_GFP_0mM_IPTG_240mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">240 minutes</div></td>
 +
    <td><div class="italic">Figure 3. Negative control: Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.</div></td>
 +
</tr>
 +
</table>
 +
</body>
 +
</html>
 +
 +
<html>
 +
<style type="text/css">
 +
#body img{
 +
    margin-bottom:3px;
 +
}
 +
table{
 +
border:2px solid black;
 +
margin-left:50px;
 +
margin-bottom: 10px;
 +
width: 650px;
 +
}
 +
.italic{
 +
font-style: italic;
 +
}
 +
 +
</style>
 +
<body>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/1/1b/BareCillus_GFP_0.05mM_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/2013/1/14/0.05mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/0/0e/BareCillus_GFP_0.05mM_IPTG_60mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">60 minutes</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/a/a6/BareCillus_GFP_0.05mM_IPTG_120mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">120 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/a/ab/BareCillus_GFP_0.05mM_IPTG_240mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">240 minutes</div></td>
 +
    <td><div class="italic">Figure 4. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.05mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.</div></td>
 +
</tr>
 +
</table>
 +
</body>
 +
</html>
 +
 +
<html>
 +
<style type="text/css">
 +
#body img{
 +
    margin-bottom:3px;
 +
}
 +
table{
 +
border:2px solid black;
 +
margin-left:50px;
 +
margin-bottom: 10px;
 +
width: 650px;
 +
}
 +
.italic{
 +
font-style: italic;
 +
}
 +
 +
</style>
 +
<body>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/b/b5/BareCillus_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/2013/8/8b/BareCillus_0.1mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/1/12/BareCillus_0.1mM_IPTG_60mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">60 minutes</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/1/1a/BareCillus_0.1mM_IPTG_120mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">120 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/f/fb/BareCillus_0.1mM_IPTG_240mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">240 minutes</div></td>
 +
    <td><div class="italic">Figure 5. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.1mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.</div></td>
 +
</tr>
 +
</table>
 +
</body>
 +
</html>
 +
 +
<html>
 +
<style type="text/css">
 +
#body img{
 +
    margin-bottom:3px;
 +
}
 +
table{
 +
border:2px solid black;
 +
margin-left:50px;
 +
margin-bottom: 10px;
 +
width: 650px;
 +
}
 +
.italic{
 +
font-style: italic;
 +
}
 +
 +
</style>
 +
<body>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/3/30/BareCillus_GFP_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/f/f8/BareCillus_GFP_0.2mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/a/a7/BareCillus_GFP_0.2mM_IPTG_60mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">60 minutes</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/2/2d/BareCillus_GFP_0.2mM_IPTG_120mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">120 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/7/76/BareCillus_GFP_0.2mM_IPTG_240mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">240 minutes</div></td>
 +
    <td><div class="italic">Figure 6. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.2mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.</div></td>
 +
</tr>
 +
</table>
 +
</body>
 +
</html>
 +
 +
<html>
 +
<style type="text/css">
 +
#body img{
 +
    margin-bottom:3px;
 +
}
 +
table{
 +
border:2px solid black;
 +
margin-left:50px;
 +
margin-bottom: 10px;
 +
width: 650px;
 +
}
 +
.italic{
 +
font-style: italic;
 +
}
 +
 +
</style>
 +
<body>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/6/6b/BareCillus_GFP_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/f/fd/BaeCillus_GFP_0.4mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/9/9d/BareCillus_GFP_0.4mM_IPTG_60mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">60 minutes</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/7/78/BareCillus_GFP_0.4mM_IPTG_120mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">120 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/d/d2/BareCillus_GFP_0.4mM_IPTG_240mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">240 minutes</div></td>
 +
    <td><div class="italic">Figure 7. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.4mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.</div></td>
 +
</tr>
 +
</table>
 +
</body>
 +
</html>
 +
 +
<html>
 +
<style type="text/css">
 +
#body img{
 +
    margin-bottom:3px;
 +
}
 +
table{
 +
border:2px solid black;
 +
margin-left:50px;
 +
margin-bottom: 10px;
 +
width: 650px;
 +
}
 +
.italic{
 +
font-style: italic;
 +
}
 +
 +
</style>
 +
<body>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/c/c6/BareCillus_GFP_0.8mM_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/c/cd/BareCillus_GFP_0.8mM_IPTG_30mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">30 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/4/4f/BareCillus_GFP_0.8mM_IPTG_60mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">60 minutes</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/a/a0/BareCillus_GFP_0.8mM_IPTG_120mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">120 minutes</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/3/35/BareCillus_GFP_0.8mM_IPTG_240mins.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">240 minutes</div></td>
 +
    <td><div class="italic">Figure 8. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.8mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.</div></td>
 +
</tr>
 +
</table>
 +
</body>
 +
</html>
  
  

Revision as of 13:42, 22 September 2013

HBsu-sfGFP

This BioBrick codes for a HBsu protein attached to a superfolded green fluorescent protein (sfGFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the sfGFP 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.

HBsu-sfGFP Testing and Characterisation

To test that HBsu-sfGFP (BBa_K1185001)BioBrick works as designed; we transformed Bacillus subtilis str. 168 with the pMUTIN4 plasmid containing our HBsu-sfGFP construct. There were colonies on Plate 1 which is LB + 5ug/ml ery plates containing the transforms B. subtilis with the plasmid containing our constructs as well as plate 2 which act as our positive control. There is 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 display in Figure 1.

Pulpit rock
Plate 1: B. subtilis transformed with HBsu-sfGFP plated on LB + ery (5ug/ml) plate.
 Pulpit rock
Plate 2: B. subtilis transformed with pGFPrrnB plated on LB + ery (5ug/ml) plate(positive control).
Pulpit rock
Plate 3: B. subtilis transformed with water plated on LB plate (viability of cells).
Figure 1. Transformation plates of ''Bacillus subtilis'' with HBsu-sfGFP, 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 were 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 green fluorescence emitted by HBsu-sfGFP at 560nm, 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-sfGFP and the right corner shows fluoresecense with DAPI staining. The HBsu-sfGFP and DAPI fluorescence shows same localisation in signal, indicating our BioBrick actually binds to DNA.

Pulpit rock
10 minutes
 Pulpit rock
30 minutes
Pulpit rock
60 minutes
 Pulpit rock
120 minutes
Pulpit rock
240 minutes
Figure 3. Negative control: Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

Pulpit rock
10 minutes
 Pulpit rock
30 minutes
Pulpit rock
60 minutes
 Pulpit rock
120 minutes
Pulpit rock
240 minutes
Figure 4. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.05mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

Pulpit rock
10 minutes
 Pulpit rock
30 minutes
Pulpit rock
60 minutes
 Pulpit rock
120 minutes
Pulpit rock
240 minutes
Figure 5. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.1mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

Pulpit rock
10 minutes
 Pulpit rock
30 minutes
Pulpit rock
60 minutes
 Pulpit rock
120 minutes
Pulpit rock
240 minutes
Figure 6. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.2mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

Pulpit rock
10 minutes
 Pulpit rock
30 minutes
Pulpit rock
60 minutes
 Pulpit rock
120 minutes
Pulpit rock
240 minutes
Figure 7. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.4mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

Pulpit rock
10 minutes
 Pulpit rock
30 minutes
Pulpit rock
60 minutes
 Pulpit rock
120 minutes
Pulpit rock
240 minutes
Figure 8. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.8mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 339