Difference between revisions of "Team:IISc-Bangalore/InterLab"

 
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         <li><a href="#introduction">Introduction<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
 
         <li><a href="#introduction">Introduction<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
 
<li><a href="#Study">InterLab Study<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
 
<li><a href="#Study">InterLab Study<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
−
<li><a href="#ludox">LUDOX<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
+
<li><a href="#standard">Standardization<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
−
<li><a href="#fluorescein-standard-curve">Fluorescein<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
+
 
<li><a href="#cell-measurements">Cell Measurements<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
 
<li><a href="#cell-measurements">Cell Measurements<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
 
<li><a href="#results">Our Results<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
 
<li><a href="#results">Our Results<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
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<div id="contentMain">
 
<div id="contentMain">
 +
 +
<img src="https://static.igem.org/mediawiki/2017/a/a1/T--IISc-Bangalore--Header--interlab.svg" id="headerImg" />
  
 
<h1 id="introduction">Introduction</h1>
 
<h1 id="introduction">Introduction</h1>
  
−
<h2 align="middle">Extraordinary claims require extraordinary evidence!</h2>
+
<h2 align="middle">Extraordinary claims require astounding evidence</h2>
 
<h3 align="middle">That which can be asserted without evidence can be dismissed without evidence...</h3>
 
<h3 align="middle">That which can be asserted without evidence can be dismissed without evidence...</h3>
  
−
<p>The sensitivity of biological systems to the environment makes it difficult to reproduce the results with sufficient accuracy. Choice of variables is of utmost importance when you want to test your model. So designing an experiment to do the same is an equally challenging task.</p>
+
<p>The sensitivity of biological systems to the environment makes it difficult to reproduce results with accuracy and designing an easily-repeatable experiment to do the same is a challenging task. The key lies in choosing variables that are both easy to measure and consistent between independent replicates.</p>
  
−
<p>Given you do the experiment and obtain some data, then you try to see the agreement between the result and the expected trends. Measurement of the fluorescence of the GFP protein is one such variable, which can be measured with a lot of precision. Though the fluorescent protein based assays are limited by folding time and other factors, these techniques have been optimized and are employed in various labs.</p>
+
<p>One such variable is the fluorescence of GFP, a quantity that can be measured rapidly with high precision. Though fluorescent protein-based assays are limited by folding time, half-life, photo-bleaching and other factors, these techniques have been optimized and are employed in various labs.</p>
  
−
<h3>InterLab Protocol</h3>
+
<h2>InterLab Protocol 2017</h2>
  
−
<p>The protocol is not only very long, but also required us to be well versed with all the lab techniques required to work with cells. All the care was taken to ensure that we minimize the possibility of false positives for instance the covering of the falcons with aluminum foil so ensure that the chloramphenicol is not degraded by light. The very minute details really helped us over the course of the interlab protocol and in our own project.</p>
+
<p>The InterLab protocol is designed to characterize the relative strengths of certain promoters and ribosome-binding sites (found in the six Test Devices) by measuring the expression of a downstream fluorescent protein (GFP) using fluorescence as a metric. The ultimate aim of the study is to develop a reliable, repeatable experiment that yields absolute units for GFP measurements using a plate reader.
  
−
<p>Also we got acquainted with the various aspects of using the plate reader. The physical concept of orbital averaging and its application in the plate reader is quite intriguing.</p>
+
<p>The protocol for this year is involved and requires us to take extreme caution to minimize the possibility of errors: for instance, our Falcons were wrapped with aluminum foil to reduce photo-bleaching of expressed GFP. Towards the goal of ensuring reproducibility, all teams this year were required to measure GFP expression using a plate reader.</p>
  
−
<p>The interlab protocol was repeated in two different labs, but due to technical issues we took the readings on the same machine, making sure that the settings were the same for all the measurements.  The understanding of the working of spectrophotometer was an important part of our project as it was one of the assays we employed to assay the floatation of the gas vesicles.</p>
 
  
−
<p>Just to be sure about the false positives, after the experiment was carried out, we saw the plates under UV transilluminator to check for the fluorescence, don’t worry we did this for the master plate and not for the re-streaked ones. They did fluorescence, saved us the trouble of checking for false positives.  [Insert the UV transilluminator images here]</p>
+
<p>On the advice of our mentor, we executed the cell measurements for the InterLab protocol twice in two different labs, but used the same plate reader and the same settings for all measurements. By performing this experiment, the principles of fluorometry and spectrophotometry became clearer to us, and this became an important part of our project as it inspired us to design a spectrophotometry-based flotation assay for gas vesicles!</p>
  
−
<h3>Why we need to replicate the experiment?</h3>
+
<p>Another simple test for fluorescence is to simply view the colonies using a UV transilluminator and check for fluorescence, which allows us to quickly confirm if a colony is expressing GFP.</p>
−
<p>In good sciences we get a multitude of unexpected and strange results. A good theory is something which can be tested repeatedly to get replicable results. This can only happen if we have correct data, for which we need to take care of false positives at every step.</p>
+
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/1/13/T--IISc-Bangalore--Fluorescentfinal.jpg" width="80%">
 +
<br>
 +
</figure>
 +
 
 +
<p>The physical concept of orbital averaging and its application in the plate reader is quite intriguing.</p>
 +
 
 +
 
 +
<h2>The Importance of Replicates</h3>
 +
<p>In scientific research, we often obtain a multitude of unexpected and strange results, prompting us to verify them. The best way to determine if these results are anomalous or indicative of an underlying causative mechanism is by repeating the experiment: the more replicates that give the same "anomalous" result, the more certain we are that we are actually correct. Our results for the InterLab study seemed anomalous at first, which made us repeat the entire experiment independently, starting from getting fresh transformants of the Test Devices. However, this independent trial yielded similar data, confirming that our results — despite their apparent oddness — are in fact correct.</p>
  
 
<h1 id="Study">InterLab Study</h1>
 
<h1 id="Study">InterLab Study</h1>
  
−
<p> Following the protocol, we took the LUDOX and Fluorescein measurements and transformed the eight interlab biobricks using E.coli DH5α competent cells (prepared by TSS method). The controls were clean and nine colonies were re-streaked on a fresh LB Plate. We used the plate 6 for all the transformations.</p>
+
<p>Following the given protocol, we took the LUDOX and fluorescein measurements and transformed <i>E.coli</i> DH5α with the eight InterLab BioBricks from Plate 6 — positive and negative controls, and six Test devices. The controls for the transformation were clean and nine colonies were re-streaked on a fresh LB Plate.</p>
 +
 
 +
<p>All the six Test devices and the positive control express GFP while the negative control is the tetR promoter, which should not produce any fluorescent protein.
  
−
<p>All the six test devices have and the positive control have built-in GFP and the negative control has Tet R which is non-fluorescent. They are all under chloramphenicol selection marker built-in a pSB1C3 vector.</p>
 
−
<ul>
 
−
<li>Positive Control (BBa_I20270)
 
 
<figure>
 
<figure>
−
<img src="https://static.igem.org/mediawiki/2017/b/b6/T--IISc-Bangalore--PlateB.jpg" width=500px>
+
<img src="https://static.igem.org/mediawiki/2017/9/91/T--IISc-Bangalore--RestreakedPlates.jpg" width=500px>
 
<br>
 
<br>
 +
<figurecaption>Nine colonies of each of the InterLab transformants were re-streaked onto a fresh plate</figurecaption>
 +
</figure>
 +
 +
<h2>Positive Control (BBa_I20270)</h2>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/6/67/T--IISc-Bangalore--interlab-positive-control.png" width="50%">
 +
</figure>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/b/b6/T--IISc-Bangalore--PlateB.jpg" width=500px>
 
<figurecaption>InterLab Plate B (Positive Control)</figurecaption>
 
<figurecaption>InterLab Plate B (Positive Control)</figurecaption>
 
</figure>
 
</figure>
−
</li>
+
 
−
<li>Negative Control (BBa_R0040)
+
<h2>Negative Control (BBa_R0040)</h2>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/d/d3/T--IISc-Bangalore--interlab-negative-control.png" width="100%">
 +
</figure>
 +
 
 
<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2017/3/37/T--IISc-Bangalore--plateD.jpg" width=500px>
 
<img src="https://static.igem.org/mediawiki/2017/3/37/T--IISc-Bangalore--plateD.jpg" width=500px>
−
<br>
 
 
<figurecaption>InterLab Plate D (Negative Control)</figurecaption>
 
<figurecaption>InterLab Plate D (Negative Control)</figurecaption>
 
</figure>
 
</figure>
−
</li>
+
 
−
<li>Test Device 1  (BBa_J364000) J23101 + I13504
+
 
 +
<h2>Test Device 1  (BBa_J364000) J23101 + I13504</h2>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/b/bf/T--IISc-Bangalore--interlab-td1.png" width="50%">
 +
</figure>
 +
 
 
<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2017/f/f8/T--IISc-Bangalore--PlateF.jpg" width=500px>
 
<img src="https://static.igem.org/mediawiki/2017/f/f8/T--IISc-Bangalore--PlateF.jpg" width=500px>
−
<br>
 
 
<figurecaption>InterLab Plate F (Test Device 1)</figurecaption>
 
<figurecaption>InterLab Plate F (Test Device 1)</figurecaption>
 
</figure>
 
</figure>
−
</li>
+
 
−
<li>Test Device 2 (BBa_J364001) J23106 + I13504
+
<h2>Test Device 2 (BBa_J364001) J23106 + I13504</h2>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/d/d2/T--IISc-Bangalore--interlab-td2.png" width="50%">
 +
</figure>
 +
 
 
<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2017/5/52/T--IISc-Bangalore--PlateH.jpg" width=500px>
 
<img src="https://static.igem.org/mediawiki/2017/5/52/T--IISc-Bangalore--PlateH.jpg" width=500px>
−
<br>
 
 
<figurecaption>InterLab Plate H (Test Device 2)</figurecaption>
 
<figurecaption>InterLab Plate H (Test Device 2)</figurecaption>
 
</figure>
 
</figure>
−
</li>
+
 
−
<li>Test Device 3 (BBa_J364002) J23117 + I13504
+
<h2>Test Device 3 (BBa_J364002) J23117 + I13504</h2>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/c/c1/T--IISc-Bangalore--interlab-td3.png" width="50%">
 +
</figure>
 +
 
 
<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2017/5/56/T--IISc-Bangalore--PlateJ.jpg" width=500px>
 
<img src="https://static.igem.org/mediawiki/2017/5/56/T--IISc-Bangalore--PlateJ.jpg" width=500px>
−
<br>
 
 
<figurecaption>InterLab Plate J (Test Device 3)</figurecaption>
 
<figurecaption>InterLab Plate J (Test Device 3)</figurecaption>
 
</figure>
 
</figure>
−
</li>
+
 
−
<li>Test Device 4 (BBa_J364003) J23101.BCD2.E0040.B0015  
+
<h2>Test Device 4 (BBa_J364003) J23101.BCD2.E0040.B0015</h2>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/7/74/T--IISc-Bangalore--interlab-td4.png" width="50%">
 +
</figure>
 +
 
 
<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2017/9/92/T--IISc-Bangalore--PlateL.jpg" width=500px>
 
<img src="https://static.igem.org/mediawiki/2017/9/92/T--IISc-Bangalore--PlateL.jpg" width=500px>
−
<br>
 
 
<figurecaption>InterLab Plate L (Test Device 4)</figurecaption>
 
<figurecaption>InterLab Plate L (Test Device 4)</figurecaption>
 
</figure>
 
</figure>
−
</li>
 
−
<h1 id="ludox">LUDOX</h1>
 
  
−
<p>To find the conversion factor of absorbance value Abs600 to optical density value OD600, the OD600 of LUDOX – S40 was used as a reference point.</p>
+
<h2>Test Device 5 (BBa_J364004) J23106.BCD2.E0040.B0015</h2>
  
−
<h1 id="fluorescein-standard-curve">Fluorescein Standardization</h1>
+
<figure>
−
<p>This curve will be used to find out the corresponding fluorescein and concentration of GFP using the cell measurements. We tried different gain values and we got the best possible results for gain ZERO i.e very few of them went out of the range. These readings were not taken again when we were replicating the cell measurements due to unavailability of the compound. Also it did not make much sense since it is a chemical and human errors or the environmental conditions are going to have less effect on them.</p>
+
<img src="https://static.igem.org/mediawiki/2017/f/fb/T--IISc-Bangalore--interlab-td5.png" width="50%">
 +
</figure>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/8/8d/T--IISc-Bangalore--PlateN.jpg" width=500px>
 +
<figurecaption>InterLab Plate N (Test Device 5)</figurecaption>
 +
</figure>
 +
 
 +
<h2>Test Device 6 (BBa_J364005) J23117.BCD2.E0040.B0015</h2>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/d/d4/T--IISc-Bangalore--interlab-td6.png" width="50%">
 +
</figure>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/9/93/T--IISc-Bangalore--PlateP.jpg" width=500px>
 +
<figurecaption>InterLab Plate P (Test Device 6)</figurecaption>
 +
</figure>
 +
 
 +
<h1 id="standard">Standardization of Fluorescence</h1>
 +
 
 +
<h2>LUDOX</h2>
 +
<p>To determine the conversion factor of absorbance value Abs600 to optical density value OD600, the OD600 of LUDOX–S40 was used as a reference point.</p>
 +
 
 +
<h2>Fluorescein Standard Curve</h2>
 +
<p>This curve will be used to determine the corresponding fluorescein and concentration of GFP during the cell measurements. We tried different gain values, and we obtained the best results for gain ZERO, i.e very few of them went out of the range.</p>
  
 
<h2>Plate reader settings</h2>
 
<h2>Plate reader settings</h2>
  
 
<ul>
 
<ul>
−
<li>Wavelength : 600 nm</li>
+
<li>Wavelength: 600 nm</li>
−
<li>Path Length Correction : OFF</li>
+
<li>Path Length Correction: OFF</li>
−
<li>Path Length Correction : OFF</li>
+
<li>Excitation: 485 nm</li>
−
<li>Excitation : 485 nm</li>
+
<li>Emission: 515 nm</li>
−
<li>Emission : 515 nm</li>
+
<li>Optics: Top</li>
−
<li>Optics : Top</li>
+
<li>Gain: ZERO</li>
−
<li>Gain : ZERO</li>
+
 
</ul>
 
</ul>
  
 
<h1 id="cell-measurements">Cell Measurements</h1>
 
<h1 id="cell-measurements">Cell Measurements</h1>
−
 
−
<p>We used E.coli DH5α cells to transform all the interlab biobricks. The protocols used for preparation of competent cells and transformation have been given.</p>
 
  
 
<h2>Discussion</h2>
 
<h2>Discussion</h2>
−
The fluorescence, OD and fluorescein/OD graphs were showing similar trends for both the colonies used. Devices 1-3 constructs are very similar to each other varying only in the promoters, similar is the case for devices 4-6.
+
<p>The fluorescence, OD, and fluorescein/OD graphs showed similar trends for both the colonies used. The Test Devices are similar constructs, varying only in the promoters, and the case is similar for devices 4-6.</p>
  
−
<h2>INSERT GRAPH</h2>
+
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/9/90/T--IISc-Bangalore--Optical_Density.png" width=500px>
 +
<br>
 +
<figurecaption>Optical density vs time</figurecaption>
 +
</figure>
  
−
<p>All the cultures seem to be growing fine, except for the Test device 1. But since the same was observed on replicating the experiment also, I think it is safe to assume that it is just taking annoyingly long time in the log phase. For the sake of our experiment it is not of much use to compare the growth of various transformants, but since the test device 1 is growing very slow, it doesn’t seem to be very useful if one intends to cell based readings to find GFP concentration. Other than that, others have fairly similar growth trends.</p>
+
<p>All the cultures grow well except for Test Device 1, but since this was observed even on replicating the experiment, it is safe to assume that it naturally grows slower than the rest. This is not particularly relevant to our experiment.</p>
  
−
<h2>INSERT GRAPH</h2>
+
<figure>
−
 
+
<img src="https://static.igem.org/mediawiki/2017/3/3e/T--IISc-Bangalore--Fluorescence.png" width=500px>
−
<p>The fluorescence readings are kind of confusing since we did not expect negative control to express any GFP, this could be the mislabeling because there was no fluorescence shown by the cells on the plate when kept under UV. If we assume this to be mislabeling, other than that Test Device 2 and Test Device 4 seem to be very efficient in the production of GFP.</p>
+
<br>
 +
<figurecaption>Fluorescence vs time</figurecaption>
 +
</figure>
  
−
<p>On the other hand if you compare the amount of GFP released per unit cell mass, Test Device 2 and Test Device 4 seem to be the best choices since they have much higher GFP production stemming from a certain concentration of cells, which trend is almost the same for all the test devices ( except test device 1). Test Device also has pretty high GFP production per unit cell mass, but we found that it is growing very slow. Though we are still unable to say anything about the negative control.<p>
+
<p>Our fluorescence readings seem anomalous since we do not expect the negative control to express any GFP. At first, we assumed this was due to mislabeling because the colonies of the negative control did not show fluorescence under UV, but our second replicate also showed such results. Apart from this error, Test Device 2 and Test Device 4 express GFP much better compared to the remaining Test Devices.</p>
  
−
<p>Since the fluorescent method seemed to be a good enough way of testing for false positives, without doing miniprep or colony PCRs, we did not bother to test the negative control.</p>
+
<p>Even when accounting for different growth rates by comparing the amount of GFP expressed per unit cell mass, Test Device 2 and Test Device 4 seem to be the best choices since they have the highest GFP productions even among other Test Devices with similar growth rates (all of them are similar except Test Device 1, which despite growing much slower has quite high GFP expression per unit cell mass).<p>
  
 
<h1 id="results">Our Results</h1>
 
<h1 id="results">Our Results</h1>
  
−
<p>It appears that promoter J23106 and J23101 seem to be elucidating strong GFP expression. Though there seems to be very less correlation in the cell proliferation and GFP production which is contrary to what I expected.</p>
+
<p>From the measured fluorescence reading, promoters J23106 and J23101 elucidate the strongest GFP expression compared to the other promoters tested.</p>
  
 
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Latest revision as of 02:25, 2 November 2017

  1. Introduction
  2. InterLab Study
  3. Standardization
  4. Cell Measurements
  5. Our Results

Introduction

Extraordinary claims require astounding evidence

That which can be asserted without evidence can be dismissed without evidence...

The sensitivity of biological systems to the environment makes it difficult to reproduce results with accuracy and designing an easily-repeatable experiment to do the same is a challenging task. The key lies in choosing variables that are both easy to measure and consistent between independent replicates.

One such variable is the fluorescence of GFP, a quantity that can be measured rapidly with high precision. Though fluorescent protein-based assays are limited by folding time, half-life, photo-bleaching and other factors, these techniques have been optimized and are employed in various labs.

InterLab Protocol 2017

The InterLab protocol is designed to characterize the relative strengths of certain promoters and ribosome-binding sites (found in the six Test Devices) by measuring the expression of a downstream fluorescent protein (GFP) using fluorescence as a metric. The ultimate aim of the study is to develop a reliable, repeatable experiment that yields absolute units for GFP measurements using a plate reader.

The protocol for this year is involved and requires us to take extreme caution to minimize the possibility of errors: for instance, our Falcons were wrapped with aluminum foil to reduce photo-bleaching of expressed GFP. Towards the goal of ensuring reproducibility, all teams this year were required to measure GFP expression using a plate reader.

On the advice of our mentor, we executed the cell measurements for the InterLab protocol twice in two different labs, but used the same plate reader and the same settings for all measurements. By performing this experiment, the principles of fluorometry and spectrophotometry became clearer to us, and this became an important part of our project as it inspired us to design a spectrophotometry-based flotation assay for gas vesicles!

Another simple test for fluorescence is to simply view the colonies using a UV transilluminator and check for fluorescence, which allows us to quickly confirm if a colony is expressing GFP.


The physical concept of orbital averaging and its application in the plate reader is quite intriguing.

The Importance of Replicates

In scientific research, we often obtain a multitude of unexpected and strange results, prompting us to verify them. The best way to determine if these results are anomalous or indicative of an underlying causative mechanism is by repeating the experiment: the more replicates that give the same "anomalous" result, the more certain we are that we are actually correct. Our results for the InterLab study seemed anomalous at first, which made us repeat the entire experiment independently, starting from getting fresh transformants of the Test Devices. However, this independent trial yielded similar data, confirming that our results — despite their apparent oddness — are in fact correct.

InterLab Study

Following the given protocol, we took the LUDOX and fluorescein measurements and transformed E.coli DH5α with the eight InterLab BioBricks from Plate 6 — positive and negative controls, and six Test devices. The controls for the transformation were clean and nine colonies were re-streaked on a fresh LB Plate.

All the six Test devices and the positive control express GFP while the negative control is the tetR promoter, which should not produce any fluorescent protein.


Nine colonies of each of the InterLab transformants were re-streaked onto a fresh plate

Positive Control (BBa_I20270)

InterLab Plate B (Positive Control)

Negative Control (BBa_R0040)

InterLab Plate D (Negative Control)

Test Device 1 (BBa_J364000) J23101 + I13504

InterLab Plate F (Test Device 1)

Test Device 2 (BBa_J364001) J23106 + I13504

InterLab Plate H (Test Device 2)

Test Device 3 (BBa_J364002) J23117 + I13504

InterLab Plate J (Test Device 3)

Test Device 4 (BBa_J364003) J23101.BCD2.E0040.B0015

InterLab Plate L (Test Device 4)

Test Device 5 (BBa_J364004) J23106.BCD2.E0040.B0015

InterLab Plate N (Test Device 5)

Test Device 6 (BBa_J364005) J23117.BCD2.E0040.B0015

InterLab Plate P (Test Device 6)

Standardization of Fluorescence

LUDOX

To determine the conversion factor of absorbance value Abs600 to optical density value OD600, the OD600 of LUDOX–S40 was used as a reference point.

Fluorescein Standard Curve

This curve will be used to determine the corresponding fluorescein and concentration of GFP during the cell measurements. We tried different gain values, and we obtained the best results for gain ZERO, i.e very few of them went out of the range.

Plate reader settings

  • Wavelength: 600 nm
  • Path Length Correction: OFF
  • Excitation: 485 nm
  • Emission: 515 nm
  • Optics: Top
  • Gain: ZERO

Cell Measurements

Discussion

The fluorescence, OD, and fluorescein/OD graphs showed similar trends for both the colonies used. The Test Devices are similar constructs, varying only in the promoters, and the case is similar for devices 4-6.


Optical density vs time

All the cultures grow well except for Test Device 1, but since this was observed even on replicating the experiment, it is safe to assume that it naturally grows slower than the rest. This is not particularly relevant to our experiment.


Fluorescence vs time

Our fluorescence readings seem anomalous since we do not expect the negative control to express any GFP. At first, we assumed this was due to mislabeling because the colonies of the negative control did not show fluorescence under UV, but our second replicate also showed such results. Apart from this error, Test Device 2 and Test Device 4 express GFP much better compared to the remaining Test Devices.

Even when accounting for different growth rates by comparing the amount of GFP expressed per unit cell mass, Test Device 2 and Test Device 4 seem to be the best choices since they have the highest GFP productions even among other Test Devices with similar growth rates (all of them are similar except Test Device 1, which despite growing much slower has quite high GFP expression per unit cell mass).

Our Results

From the measured fluorescence reading, promoters J23106 and J23101 elucidate the strongest GFP expression compared to the other promoters tested.