Difference between revisions of "Part:BBa K523014"

(→‎Experiment)
 
(27 intermediate revisions by 3 users not shown)
Line 8: Line 8:
 
The BglX β-glucosidase is expected to be localised to the periplasm. It ought to be capable of breaking down cellobiose.
 
The BglX β-glucosidase is expected to be localised to the periplasm. It ought to be capable of breaking down cellobiose.
  
−
====Experiment====
+
====MUG/MUC experiment====
  
−
We conducted two assays, comparing the activity of this part ''(bglX)'' with that of the exoglucanase cex (<partinfo>BBa_K118022</partinfo>) on two different substrates:
+
We (Edinburgh 2011) conducted two assays, comparing the activity of this part ''(Plac-bglX)'' with that of the exoglucanase ''cex'' under the control of the lac promoter (<partinfo>BBa_K523016</partinfo>) on two different substrates:
  
−
* 4-methylumbelliferyl-beta-D-glucuronide '''(MUG, left photo)'''. This substrate mimics cellobiose but produces a fluorescent product when cleaved.
+
* 4-methylumbelliferyl &beta;- D- glucuronide '''(MUG, left photo)'''. This substrate is a cellobiose analog.
 +
* 4-methylumbelliferyl &beta;- D- cellobioside '''(MUC, right photo)'''. This substrate is larger and is more like a cellulose analog.
  
−
* Something else '''(MUC, right photo)'''.
+
Both substrates produce a fluorescent product when cleaved. Our plates below show the results of placing cell lysate and cell debris on an MUG plate and an MUC plate. Present on both plates are:
  
−
Our plates below shows the results of placing cell lysate and cell debris on an MUG plate. Present on both plates are:
+
* Left side of plate: lysate/debris from JM109 expressing this part, K523014
−
 
+
* Right side of plate: lysate/debris from JM109 expressing exoglucanase ''cex'', <partinfo>BBa_K523016</partinfo>
−
* Left side of plate: JM109 expressing this part, K523014
+
* Bottom of plate: lysate/debris from JM109 cells
−
* Right side of plate: JM109 expressing exoglucanase
+
−
* Bottom of plate: JM109 cells
+
  
 
<center>
 
<center>
Line 29: Line 28:
 
|[[Image:Cex-MuC.jpg|300px]]
 
|[[Image:Cex-MuC.jpg|300px]]
 
|-
 
|-
−
|width="300px" valign="top"|Foo.
+
|width="300px" valign="top"|'''MUG assay.''' ''bglX'' on left, ''cex'' on right.
 
|&nbsp; &nbsp;
 
|&nbsp; &nbsp;
−
|width="300px" valign="top"|Bar.
+
|width="300px" valign="top"|'''MUC assay.''' ''bglX'' on left, ''cex'' on right.
 
|}
 
|}
 
</center>
 
</center>
 +
 +
As can be seen, ''bglX'' is capable of degrading MUG (the cellobiose analog) while exoglucanase displays much weaker activity. By contrast, exoglucanase is much better at degrading MUC.
 +
 +
====Inability to grow on cellobiose====
 +
 +
We tested the ability of ''E. coli'' with this part to grow on minimal media with cellobiose as the only carbon source. It could not. By contrast, it did grow if glucose was the carbon source, showing that it is fundamentally capable of growing on minimal media.
 +
 +
 +
<center>
 +
{| style="margin-top: 1em; margin-bottom: 1em;"
 +
|-
 +
|[[Image:K523014 on cellobiose.jpg|300px]]
 +
|&nbsp; &nbsp;
 +
|[[Image:K523014 glucose control.jpg|300px]]
 +
|-
 +
|width="300px" valign="top"|Cellobiose as the only carbon source. K523014 (bottom) fails to grow.
 +
|&nbsp; &nbsp;
 +
|width="300px" valign="top"|Glucose as the only carbon source. K523014 can grow.
 +
|}
 +
</center>
 +
 +
===''E.coli'' in fluid media===
 +
(characterize by SDU-Denmark)
 +
 +
It was not possible to see any indications of ''E.coli'' being capable of living on cellobiose in fluid media. On the figure below, it is shown that this biobrick can not make ''E.coli'' grow with cellobiose as the only carbon source. The bglX is compared to another biobrick which have the ability to degrade cellobiose to glucose cep94A/<partinfo>BBa_K2449004</partinfo>
 +
 +
<center>
 +
https://static.igem.org/mediawiki/2017/b/b8/T--SDU-Denmark--Registry-bglX.jpg
 +
</center>
 +
<font size="2" style="text-align:center;"><b>Figure 3:</b>Growth on cellubiose comparing BBa_523014/bglX to BBa_2449004/cep94A compared</font>
 +
 +
===Conclusion===
 +
This biobrick does not work as intended.
  
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K523014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K523014 SequenceAndFeatures</partinfo>
  
  
−
<!-- Uncomment this to enable Functional Parameter display
+
 
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K523014 parameters</partinfo>
 
<partinfo>BBa_K523014 parameters</partinfo>
−
<!-- -->
+
 
 +
 
 +
 
 +
 
 +
==Functional Parameters: Austin_UTexas==
 +
<html>
 +
<body>
 +
<h3><center>Burden Imposed by this Part:</center></h3>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.org/mediawiki/parts/7/7a/T--Austin_Utexas--high_significant_burden.png" style = "width:250px;height:120px"></center>
 +
</div>
 +
<figcaption><center><b>Burden Value: 39.6 ± 4.7% </b></center></figcaption>
 +
</figure>
 +
<p> Burden is the percent reduction in the growth rate of <i>E. coli</i> cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the
 +
<a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the <a href="https://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team.</a></p>
 +
<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
 +
  </p>
 +
</body>
 +
</html>

Latest revision as of 01:05, 4 September 2020

Plac + LacZ + bglX

The E. coli periplasmic β-glucosidase gene bglX under the control of the lac promoter. The native ribosome binding site is present.

Usage and Biology

The BglX β-glucosidase is expected to be localised to the periplasm. It ought to be capable of breaking down cellobiose.

MUG/MUC experiment

We (Edinburgh 2011) conducted two assays, comparing the activity of this part (Plac-bglX) with that of the exoglucanase cex under the control of the lac promoter (BBa_K523016) on two different substrates:

  • 4-methylumbelliferyl β- D- glucuronide (MUG, left photo). This substrate is a cellobiose analog.
  • 4-methylumbelliferyl β- D- cellobioside (MUC, right photo). This substrate is larger and is more like a cellulose analog.

Both substrates produce a fluorescent product when cleaved. Our plates below show the results of placing cell lysate and cell debris on an MUG plate and an MUC plate. Present on both plates are:

  • Left side of plate: lysate/debris from JM109 expressing this part, K523014
  • Right side of plate: lysate/debris from JM109 expressing exoglucanase cex, BBa_K523016
  • Bottom of plate: lysate/debris from JM109 cells
BglX-MuG.jpg     Cex-MuC.jpg
MUG assay. bglX on left, cex on right.     MUC assay. bglX on left, cex on right.

As can be seen, bglX is capable of degrading MUG (the cellobiose analog) while exoglucanase displays much weaker activity. By contrast, exoglucanase is much better at degrading MUC.

Inability to grow on cellobiose

We tested the ability of E. coli with this part to grow on minimal media with cellobiose as the only carbon source. It could not. By contrast, it did grow if glucose was the carbon source, showing that it is fundamentally capable of growing on minimal media.


K523014 on cellobiose.jpg     K523014 glucose control.jpg
Cellobiose as the only carbon source. K523014 (bottom) fails to grow.     Glucose as the only carbon source. K523014 can grow.

E.coli in fluid media

(characterize by SDU-Denmark)

It was not possible to see any indications of E.coli being capable of living on cellobiose in fluid media. On the figure below, it is shown that this biobrick can not make E.coli grow with cellobiose as the only carbon source. The bglX is compared to another biobrick which have the ability to degrade cellobiose to glucose cep94A/BBa_K2449004

T--SDU-Denmark--Registry-bglX.jpg

Figure 3:Growth on cellubiose comparing BBa_523014/bglX to BBa_2449004/cep94A compared

Conclusion

This biobrick does not work as intended.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 607
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2266
    Illegal AgeI site found at 2488
    Illegal AgeI site found at 2677
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters



Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden Value: 39.6 ± 4.7%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.