Difference between revisions of "Team:Vilnius-Lithuania-OG/InterLab"

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<h1>InterLab</h1>
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<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study and/or obtain new, high quality experimental characterization data for an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2018 part number range.
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<title>Collaborations</title>
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<br><br>
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<h2 data-caption-animate="fadeInUp">Interlab</h2>
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<li><a href="#" data-href="#overview">Overview</a></li>
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<li><a href="#" data-href="#section-methods">Methods and Results</a></li>
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 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">The
 +
fifth InterLab study has launched and it further attempts to develop
 +
a standard procedure for green fluorescent protein (GFP) measurements
 +
to reduce variability across laboratories in the world. This year the
 +
attempt is to normalize fluorescence measurements to colony forming
 +
units (CFU) instead of optical density of the culture since it is a
 +
great source of variability among labs. Two approaches were used to
 +
compute a cell number in the samples: </font>
 +
</p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 100%"><br/></p>
 +
 
 +
<ol style="PADDING-LEFT: 30px">
 +
<li/>
 +
<p style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">Converting
 +
absorbance of cells to absorbance of known concentration beads;</font></p>
 +
<li/>
 +
<p style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">Counting
 +
CFU in the samples. </font>
 +
</p>
 +
</ol>
 +
 
 +
<div class="divider divider-center"><i class="icon-circle"></i></div>
 +
 
 +
<section id="section-methods" class="page-section">
 +
 
 +
</p>
 +
<h2>Methods and Results</h2>
 +
<div class="heading-block left"></div>
 +
 
 +
 +
<div class="fancy-title title-border-color">
 +
<h4>1. Calibration protocols</h4>
 +
</div>
 +
 
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">At
 +
first, we measured absorbance (Abs<sub>600</sub>)
 +
of weakly scattering LUDOX CL-X to obtain a multiplication factor of
 +
Abs<sub>600</sub>
 +
to transform absorbance values to the comparable OD<sub>600</sub>
 +
units. </font>
 +
</p>
 +
<p style="margin-bottom: 0in; line-height: 200%"><br/>
 +
 
 +
</p>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">Following
 +
that, we prepared serial dilutions of monodisperse silica
 +
microspheres and obtained standard particle curve (<b>Fig.
 +
1</b>) which allows to
 +
convert Abs<sub>600</sub>>measurements to
 +
an estimated number of cells. </font>
 +
</p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 300%"><br/></p>
 +
 
 +
<div align="center"><img src="/images/interlab/fig1a.png" name="fig1a"></div>
 +
<div align="center"><img src="/images/interlab/fig1b.png" name="fig1b"></div>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><center> <b> Figure
 +
1.</b> Standard particle curve obtained by using microspheres that
 +
are similar size to a cell displayed in (<b>A</b>) a linear scale and
 +
(<b>B</b>) a log scale. </p> </center></p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 100%"><br/></p>
 +
 
 +
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">The
 +
final calibration was that of fluorescein concentration. We generated
 +
a standard curve of fluorescence for fluorescein concentration by,
 +
again, making serial dilutions of fluorescein (<b>Fig.
 +
2</b>). This way we
 +
could convert our cell based readings of fluorescence to an
 +
equivalent fluorescein concentration.  The measurements were taken
 +
with the same setting as those which would be used measuring cell
 +
fluorescence. </font>
 +
</p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 300%"><br/></p>
 +
 
 +
<div align="center"><img src="/images/interlab/fig2a.png" name="fig1a"></div>
 +
<div align="center"><img src="/images/interlab/fig2b.png" name="fig1b"></div>
 +
 
 +
 
 +
<p style="line-height: 200%"><b><center> Figure 2.</b>
 +
Fluorescein standard curve obtained by serial dilutions in (<b>A</b>)
 +
the linear scale and in (<b>B</b>) the log scale. </center></p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><br/>
 +
 
 +
</p>
 +
<div class="fancy-title title-border-color">
 +
<h4>2. Cell measurement protocol</h4>
 +
</div>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">We
 +
were provided with four different devices (Test device 1 -
 +
</font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_J364000"><font size="3" style="font-size: 12pt">BBa_J364000</font></a></font><font size="3" style="font-size: 12pt">,
 +
Test device 2 - </font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_J364001"><font size="3" style="font-size: 12pt">BBa_J364001</font></a></font><font size="3" style="font-size: 12pt">,
 +
Test device 3 - </font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_J364002"><font size="3" style="font-size: 12pt">BBa_J364002</font></a></font><font size="3" style="font-size: 12pt">,
 +
Test device 4 - </font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_J364007"><font size="3" style="font-size: 12pt">BBa_J364007</font></a></font><font size="3" style="font-size: 12pt">,
 +
Test device 5 - </font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_J364008"><font size="3" style="font-size: 12pt">BBa_J364008</font></a></font><font size="3" style="font-size: 12pt">,
 +
Test device 6 - </font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_J364009"><font size="3" style="font-size: 12pt">BBa_J364009</font></a></font><font size="3" style="font-size: 12pt">)</font><font size="3" style="font-size: 12pt">
 +
along with negative (</font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_R0040"><font size="3" style="font-size: 12pt">BBa_R0040</font></a></font><font size="3" style="font-size: 12pt">)
 +
</font><font size="3" style="font-size: 12pt">and positive
 +
(</font><font color="#0000ff"><a href="http://parts.igem.org/Part:BBa_I20270"><font size="3" style="font-size: 12pt">BBa_I20270</font></a></font><font size="3" style="font-size: 12pt">)</font><font size="3" style="font-size: 12pt">
 +
controls which then we transformed into </font><font size="3" style="font-size: 12pt"><i>E.
 +
coli</i></font><font size="3" style="font-size: 12pt"> DH5</font><font size="3" style="font-size: 12pt">α</font><font size="3" style="font-size: 12pt">
 +
strain. Devices express GFP under Anderson promoters of different
 +
strength and are in the pSB1C3 backbone which carries resistance to
 +
chloramphenicol. </font>
 +
</p>
 +
<p style="margin-bottom: 0in; line-height: 200%"><br/>
 +
 
 +
</p>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">We
 +
made overnight cultures of two colonies of each transformation plate.
 +
The next day, the cultures were diluted to OD<sub>600</sub>=0.02
 +
and grown for 6 hours. The absorbance (600 nm) (<b>Fig. 3A</b>) and
 +
fluorescence (485/520, gain = 50) (<b>Fig. 3B</b>) of cultures were measured
 +
at 0 and 6 hours. </font>
 +
</p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 300%"><br/></p>
 +
 
 +
<div align="center"><img src="/images/interlab/fig3a.png" name="fig3a"></div>
 +
<div align="center"><img src="/images/interlab/fig3b.png" name="fig3b"></div>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><br/></p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><b><center> Figure 3.</b>
 +
Average absorbance (<b>A</b>) and fluorescence (<b>B</b>) measurements of the 2 colonies and 4 replicates for each
 +
sample, taken after 6 hours of growth. </center></p>
 +
 
 +
</p>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">The
 +
negative control demonstrates the highest absorbance which could be
 +
the result of the lowest metabolic load of all the devices, since it
 +
has no GFP expressed. On the other hand, devices 1, 4 and 5 show much
 +
slower bacteria growth even though the fluorescence of these devices
 +
is not the highest. Furthermore, device 1 demonstrates a really
 +
moderate absorbance and fluorescence increase after 6 hours
 +
suggesting that environmental factors could have impeded sample
 +
growth.</font></p>
 +
<p style="margin-bottom: 0in; line-height: 200%"><br/>
 +
 
 +
</p>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt"><span lang="lt-LT">Next,
 +
the previous data (absorbance and fluorescence) was normalized to the
 +
comparable OD units (<b>Fig. 4A</b>) and then to particles (<b>Fig. 4B</b>) so that we could
 +
determine the mean expression level of GFP per cell. </font>
 +
</p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 300%"><br/></p>
 +
 
 +
<div align="center"><img src="/images/interlab/fig4a.png" name="fig3a"></div>
 +
<div align="center"><img src="/images/interlab/fig4b.png" name="fig3b"></div>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><br/></p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><b><center> Figure 4.</b>
 +
Normalization of fluorescence to the OD units (<b>A</b>) and MFEL per particle (<b>B</b>). </center></p>
 +
 
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt"><span lang="lt-LT">After
 +
6 hours all the devices (except the negative control) demonstrates a
 +
relatively decreased level of fluorescence. That could be explained
 +
by cells reaching a certain level of GFP expression after which the
 +
fluorescence stops increasing whereas absorption steadily goes up.</span></font></p>
 +
<p lang="lt-LT" style="margin-bottom: 0in; line-height: 200%"><br/>
 +
 
 +
</p>
 +
 
 +
<div class="fancy-title title-border-color">
 +
<h4>3. Colony forming units (CFU) per 0.1 OD<sub>600</sub></h4>
 +
</div>
 +
 
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt"><span lang="lt-LT">The
 +
second approach is to calibrate OD</span></font><sub><font size="3" style="font-size: 12pt"><span lang="lt-LT">600</span></font></sub><font size="3" style="font-size: 12pt"><span lang="lt-LT">
 +
to CFU which reflects the cell count in the sample. For this approach
 +
only negative and positive controls were used.  </span></font>
 +
</p>
 +
<p lang="lt-LT" style="margin-bottom: 0in; line-height: 200%"><br/>
 +
 
 +
</p>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt"><span lang="lt-LT">The
 +
overnight cultures of two colonies per control were diluted to
 +
OD</span></font><sub><font size="3" style="font-size: 12pt"><span lang="lt-LT">600</span></font></sub><font size="3" style="font-size: 12pt"><span lang="lt-LT">=0.1
 +
(three replications) and serial dilutions were made which then were
 +
spreaded on agar plates. The next morning the colonies were counted
 +
to find out the number of cells in the samples (<b>Fig. 5</b>). </span></font>
 +
</p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 300%"><br/></p>
 +
 
 +
<div align="center"><img src="/images/interlab/fig5.png" name="fig3a"></div>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><br/></p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 200%"><b><center> Figure 5.</b>
 +
Normalization of fluorescence to the OD units (<b>A</b>) and MFEL per particle (<b>B</b>). </center></p>
 +
 
 +
</p>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt"><span lang="lt-LT">NC
 +
1.1 and PC 1.2 samples show relatively high and low values
 +
resspectively comparing to other samples which could indicate a
 +
systematic error in a serial dilution step. </span></font>
 +
</p>
 +
 
 +
<p style="margin-bottom: 0in; line-height: 300%"><br/></p>
 +
 
 +
<section id="section-conclusions" class="page-section">
 +
 
 +
<h2>Conclusions</h2>
 +
<div class="heading-block left"></div>
 +
<p align="justify" style="margin-bottom: 0in; line-height: 200%"><font size="3" style="font-size: 12pt">
 +
In this study cell fluorescence depends on the strength of the promotor under which GFP was expressed. However, all the cells with different devices demonstrated a reduced fluorescence per cell after 6 hours indicating a slowed down GFP production. </font></p>
 +
 
 +
 
 +
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Revision as of 23:11, 16 October 2018

Collaborations

Interlab

Explore INTERLAB

The fifth InterLab study has launched and it further attempts to develop a standard procedure for green fluorescent protein (GFP) measurements to reduce variability across laboratories in the world. This year the attempt is to normalize fluorescence measurements to colony forming units (CFU) instead of optical density of the culture since it is a great source of variability among labs. Two approaches were used to compute a cell number in the samples:


  1. Converting absorbance of cells to absorbance of known concentration beads;

  2. Counting CFU in the samples.

Methods and Results

1. Calibration protocols

At first, we measured absorbance (Abs600) of weakly scattering LUDOX CL-X to obtain a multiplication factor of Abs600 to transform absorbance values to the comparable OD600 units.


Following that, we prepared serial dilutions of monodisperse silica microspheres and obtained standard particle curve (Fig. 1) which allows to convert Abs600>measurements to an estimated number of cells.


Figure 1. Standard particle curve obtained by using microspheres that are similar size to a cell displayed in (A) a linear scale and (B) a log scale.


The final calibration was that of fluorescein concentration. We generated a standard curve of fluorescence for fluorescein concentration by, again, making serial dilutions of fluorescein (Fig. 2). This way we could convert our cell based readings of fluorescence to an equivalent fluorescein concentration. The measurements were taken with the same setting as those which would be used measuring cell fluorescence.


Figure 2. Fluorescein standard curve obtained by serial dilutions in (A) the linear scale and in (B) the log scale.


2. Cell measurement protocol

We were provided with four different devices (Test device 1 - BBa_J364000, Test device 2 - BBa_J364001, Test device 3 - BBa_J364002, Test device 4 - BBa_J364007, Test device 5 - BBa_J364008, Test device 6 - BBa_J364009) along with negative (BBa_R0040) and positive (BBa_I20270) controls which then we transformed into E. coli DH5α strain. Devices express GFP under Anderson promoters of different strength and are in the pSB1C3 backbone which carries resistance to chloramphenicol.


We made overnight cultures of two colonies of each transformation plate. The next day, the cultures were diluted to OD600=0.02 and grown for 6 hours. The absorbance (600 nm) (Fig. 3A) and fluorescence (485/520, gain = 50) (Fig. 3B) of cultures were measured at 0 and 6 hours.



Figure 3. Average absorbance (A) and fluorescence (B) measurements of the 2 colonies and 4 replicates for each sample, taken after 6 hours of growth.

The negative control demonstrates the highest absorbance which could be the result of the lowest metabolic load of all the devices, since it has no GFP expressed. On the other hand, devices 1, 4 and 5 show much slower bacteria growth even though the fluorescence of these devices is not the highest. Furthermore, device 1 demonstrates a really moderate absorbance and fluorescence increase after 6 hours suggesting that environmental factors could have impeded sample growth.


Next, the previous data (absorbance and fluorescence) was normalized to the comparable OD units (Fig. 4A) and then to particles (Fig. 4B) so that we could determine the mean expression level of GFP per cell.



Figure 4. Normalization of fluorescence to the OD units (A) and MFEL per particle (B).

After 6 hours all the devices (except the negative control) demonstrates a relatively decreased level of fluorescence. That could be explained by cells reaching a certain level of GFP expression after which the fluorescence stops increasing whereas absorption steadily goes up.


3. Colony forming units (CFU) per 0.1 OD600

The second approach is to calibrate OD600 to CFU which reflects the cell count in the sample. For this approach only negative and positive controls were used.


The overnight cultures of two colonies per control were diluted to OD600=0.1 (three replications) and serial dilutions were made which then were spreaded on agar plates. The next morning the colonies were counted to find out the number of cells in the samples (Fig. 5).



Figure 5. Normalization of fluorescence to the OD units (A) and MFEL per particle (B).

NC 1.1 and PC 1.2 samples show relatively high and low values resspectively comparing to other samples which could indicate a systematic error in a serial dilution step.


Conclusions

In this study cell fluorescence depends on the strength of the promotor under which GFP was expressed. However, all the cells with different devices demonstrated a reduced fluorescence per cell after 6 hours indicating a slowed down GFP production.