Difference between revisions of "Team:TecCEM/InterLab"

Line 24: Line 24:
 
             </li>
 
             </li>
 
             <li>
 
             <li>
−
                 <a data-target="#competent-cell"></a>Competent cell - transformation DH5-alpha</a>
+
                 <a data-target="#competentcell"></a>Competent cell - transformation DH5-alpha</a>
 
             </li>
 
             </li>
 
             <li>
 
             <li>
Line 60: Line 60:
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
−
             <div class="row offset" id="competent-cell">
+
             <div class="row offset" id="competentcell">
 
                 <div class="col">
 
                 <div class="col">
−
                     <h2>
+
                     <h1>
 
                         Competent cell - transformation DH5-alpha
 
                         Competent cell - transformation DH5-alpha
−
                     </h2>
+
                     </h1>
 
                     <p>Transformations of all devices were performed, and the resulting transformant strains were
 
                     <p>Transformations of all devices were performed, and the resulting transformant strains were
 
                         plated on LB
 
                         plated on LB
Line 74: Line 74:
 
             <div class="row offset" id="verification">
 
             <div class="row offset" id="verification">
 
                 <div class="col">
 
                 <div class="col">
−
                     <h2>Verification</h2>
+
                     <h1>Verification</h1>
 
                     <p>We verified the presence and size of the device plasmids in the transformant strains by running
 
                     <p>We verified the presence and size of the device plasmids in the transformant strains by running
 
                         an agarose gel electrophoresis.
 
                         an agarose gel electrophoresis.
Line 136: Line 136:
 
             <div class="row offset" id="calibration">
 
             <div class="row offset" id="calibration">
 
                 <div class="col">
 
                 <div class="col">
−
                     <h2>Calibration Protocol
+
                     <h1>Calibration Protocol
−
                     </h2>
+
                     </h1>
 
                     <p>
 
                     <p>
 
                         The main objective of this calibration LUDOX protocol is to use LUDOX CL-X to get the
 
                         The main objective of this calibration LUDOX protocol is to use LUDOX CL-X to get the
Line 151: Line 151:
 
                                 <th> </th>
 
                                 <th> </th>
 
                                 <th>LUDOX CL-X</th>
 
                                 <th>LUDOX CL-X</th>
−
                                 <th>H2O</th>
+
                                 <th>h1O</th>
 
                             </tr>
 
                             </tr>
 
                         </thead>
 
                         </thead>
Line 199: Line 199:
 
             <div class="row offset" id="particle">
 
             <div class="row offset" id="particle">
 
                 <div class="col">
 
                 <div class="col">
−
                     <h2>Particle standard curve - Microsphere Protocol</h2>
+
                     <h1>Particle standard curve - Microsphere Protocol</h1>
 
                     <p>The aim of this protocol is to establish a standard curve from a microsphere solution. These
 
                     <p>The aim of this protocol is to establish a standard curve from a microsphere solution. These
 
                         microspheres have similar size and optic properties to bacterial cells, therefore, they may be
 
                         microspheres have similar size and optic properties to bacterial cells, therefore, they may be
Line 232: Line 232:
 
             <div class="row offset text-center" id="fluorescence">
 
             <div class="row offset text-center" id="fluorescence">
 
                 <div class="col">
 
                 <div class="col">
−
                     <h2>Fluorescence standard curve - Fluorescein Protocol</h2>
+
                     <h1>Fluorescence standard curve - Fluorescein Protocol</h1>
 
                     <p>
 
                     <p>
 
                         Following the protocol for the fluorescence standard curve using fluorescein, we prepared the
 
                         Following the protocol for the fluorescence standard curve using fluorescein, we prepared the
Line 247: Line 247:
 
             <div class="row offset" id="cell-measurement">
 
             <div class="row offset" id="cell-measurement">
 
                 <div class="col">
 
                 <div class="col">
−
                     <h2>
+
                     <h1>
 
                         Cell measurement Protocol
 
                         Cell measurement Protocol
−
                     </h2>
+
                     </h1>
 
                     <p><strong>Table 3.</strong> Absorbance and fluorescence at hour 0 and hour 6.
 
                     <p><strong>Table 3.</strong> Absorbance and fluorescence at hour 0 and hour 6.
 
                     </p>
 
                     </p>
Line 349: Line 349:
 
             <div class="row offset" id="colony-forming">
 
             <div class="row offset" id="colony-forming">
 
                 <div class="col">
 
                 <div class="col">
−
                     <h2>Colony Forming Units per 0.1 OD<sub>600</sub> E. coli cultures
+
                     <h1>Colony Forming Units per 0.1 OD<sub>600</sub> E. coli cultures
−
                     </h2>
+
                     </h1>
 
                     <p>To preparing the starting samples we diluted overnight cultures of both positive and negative
 
                     <p>To preparing the starting samples we diluted overnight cultures of both positive and negative
 
                         controls
 
                         controls

Revision as of 16:05, 17 October 2018

Cell Gif

Interlab

Synthetic biology and all the engineering disciplines need to be reliable and repeatable, this year iGEM TecCEM 2018 is one of the teams that participated in the Fifth International InterLaboratory Measurement Study. In this page, we report the results obtained during all protocols.

Competent cell - transformation DH5-alpha

Transformations of all devices were performed, and the resulting transformant strains were plated on LB agar, with 35 μg/mL chloramphenicol. Two colonies per plate were separated into two independent cultures.

Verification

We verified the presence and size of the device plasmids in the transformant strains by running an agarose gel electrophoresis.

Table 1. Devices from the Interlab Study with their pait number and respective size.

Device Part Number Size (bp)
Negative Control BBa_R0040 2124
Positive Control BBa_I20270 2989
Device 1 BBa_J364000 2988
Device 2 BBa_J364001 2988
Device 3 BBa_J364002 2988
Device 4 BBa_J364007 2988
Device 5 BBa_J364008 2988
Device 6 BBa_J364009 2988

Calibration Protocol

The main objective of this calibration LUDOX protocol is to use LUDOX CL-X to get the conversion factor between absorbance (Abs 600) from the plate reader and the OD600 measurement.

Table 2. OD600 Reference Point - LUDOX Protocol.

LUDOX CL-X h1O
Replicate 1 0.059 0.033
Replicate 2 0.061 0.033
Replicate 3 0.062 0.033
Replicate 4 0.060 0.033
Arith. Mean 0.061 0.033
Corrected Abs600 0.028
Reference OD600 0.063
OD600/Abs600 2.291

Particle standard curve - Microsphere Protocol

The aim of this protocol is to establish a standard curve from a microsphere solution. These microspheres have similar size and optic properties to bacterial cells, therefore, they may be used to calibrate.

After preparing the Microsphere Stock Solution, serial dilutions were made in a 96-well plate as instructed. To achieve reliable results, the plate reader was set to shake the samples just before reading them. The reads were registered in the given Excel file and are shown below.

Interlab plot 1
Interlab plot 2
Figure 1. Standard curve for particle count/100 uL

Fluorescence standard curve - Fluorescein Protocol

Following the protocol for the fluorescence standard curve using fluorescein, we prepared the corresponding reactants and the requested serial solutions in the 96-well plate.

Interlab plot 3
Figure 2. Standard curve for fluorescein concentration

Cell measurement Protocol

Table 3. Absorbance and fluorescence at hour 0 and hour 6.

Hour 0 Hour 6
Abs600 Raw Readings Fluorescence Raw Readings Abs600 Raw Readings Fluorescence Raw Readings
Negative Control 0.078375 153.5 0.46225 170.125
Positive Control 0.074 220.125 0.428125 1926.75
Device 1 0.0715 439.125 0.241 4319.5
Device 2 0.079125 266.625 0.442375 2148.5
Device 3 0.075625 151.125 0.430375 207.625
Device 4 0.074375 601.5 0.347625 5545.875
Device 5 0.075625 335.875 0.16425 2039
Device 6 0.077 227.125 0.45325 1151
LB + Chlor (Blank) 0.053375 158.625 0.051125 177.5

After performing all three calibration measurements, we proceeded to execute the cell measurement protocol, previously having the overnight cell culture of each device and control. We use the same plate and volume that we used in the calibration protocol, also the same settings in order to make valid the measurement.

We defined the difference between the measurement of the absorbance and fluorescence, starting in the hour 0 until the hour 6.

Colony Forming Units per 0.1 OD600 E. coli cultures

To preparing the starting samples we diluted overnight cultures of both positive and negative controls to the linear detection range of the plate reader. A duplicate of each culture was placed on the 96-well plate, as well as a blank. After measuring absorbance, cultures were diluted further to reach an OD600=0.1. A triplicate of each dilution was placed as instructed. The absorbance measurements were confirmed and used to prepare the respective dilutions for 12 starting samples at 0.1, six for each control. We diluted the cultures to a final OD600 of 0.1 and performed serial dilutions by transferring 100 μL to the next tube. The dilutions 3, 4, and 5 were used to spread 100 μL in LB+Cam plates. After an overnight incubation, we counted the colonies in each of the plates, being many of them uncountable. The counted colonies are shown below.

Table 4. Colony count.

Plate Colony count
Dilution 3 Dilution 4 Dilution 5
A-1 564 417 49
A-2 1120 286 64
A-3 1624 360 78
B-1 984 349 53
B-2 1224 277 55
B-3 1156 369 45
A+1 1340 90 5
A+2 812 243 16
A+3 1052 71 14
B+1 1146 180 25
B+2 1116 250 21
B+3 888 138 36