Difference between revisions of "Team:SDU CHINA/InterLab"

Revision as of 06:59, 22 October 2017

Background

“All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which aimed to be one of the labs to repeat measurements in different condition. In our case, we measured fluorescence using plate reader.

Design

We often used fluorescence as an indicator of promoter activity. However, the ability to repeat measurements in different labs has been difficult. To solve this problem, InterLab study has been developed as a robust measurement procedure for green fluorescent protein (GFP) which is one of the most used markers in synthetic biology.

This year's iGEM Interlab study is to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm and GFP fluorescence for GFP expression per cell for all devices.

Materials

Plasmids used

Positive Control Device: I20270 in pSB1C3 (Located in Kit Plate 6, well 20B)
Negative Control Device: R0040 in pSB1C3 (Located in Kit Plate 6, well 20D)
Test Device 1: J23101+I13504 in pSB1C3 (Located in Kit Plate 6, well 20F)
Test Device 2: J23106+I13504 in pSB1C3 (Located in Kit Plate 6, well 20H)
Test Device 3: J23117+I13504 in pSB1C3 (Located in Kit Plate 6, well 20J)
Test Device 4: J23101.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20L)
Test Device 5: J23106.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20N)
Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)

Plasmids used

Escherichia coli strain DH5α
Materials
1ml LUDOX
H20
96 well plate
fluorescein
10ml 1xPBS
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)
50 ml Falcon tube
Incubator at 37°C
1.5 ml eppendorf tubes
Ice bucket with ice
Pipettes

Plasmids used

Thermo Multiskan FC
BERTHOLD TECHNOLOGIES LB940Mith

Protocol

Results

Sequencing

Positive Control ：BBa_I20270
Negative Control ：BBa_R0040
Test Device 1 ：BBa_J364000
Test Device 2 ：BBa_J364001
Test Device 3 ：BBa_J364002
Test Device 4 ：BBa_J364003
Test Device 5 ：BBa_J364004
Test Device 6 ：BBa_J364005

>BBa_I20270 Part-only sequence (919 bp)

ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtccc aattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattt atttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaac agcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaa gtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataac tcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcag accattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacga aaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatca aataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggt gggcctttctgcgtttata

>BBa_R0040 Part-only sequence (54 bp)

tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac

>BBa_J364000 Part-only sequence (918 bp)

tttacagctagctcagtcctaggtattatgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta tttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaaca gcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaag tttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcaga ccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaa aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg ggcctttctgcgtttata

>BBa_J364001 Part-only sequence (918 bp)

tttacggctagctcagtcctaggtatagtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta tttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaaca gcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaag tttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcaga ccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaa aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg ggcctttctgcgtttata

>BBa_J364002 Part-only sequence (918 bp)

ttgacagctagctcagtcctagggattgtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta tttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaaca gcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaag tttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcaga ccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaa aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg ggcctttctgcgtttata

>BBa_J364003 Part-only sequence (990 bp)

tttacagctagctcagtcctaggtattatgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta atgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttcc atggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgccc gaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaa tcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaa acaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggc gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

>BBa_J364004 Part-only sequence (990 bp)

tttacggctagctcagtcctaggtatagtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta atgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttcc atggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgccc gaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaa tcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaa acaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggc gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

>BBa_J364005 Part-only sequence (990 bp)

ttgacagctagctcagtcctagggattgtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta atgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttcc atggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgccc gaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaa tcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaa acaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggc gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

Data

OD600 Reference Point

Table 1. OD600 Reference Point.

Fluorescein Standard Curve

Table 2. Data of Fluorescein Standard Curve.

Figure 1

Figure 2

Normalization

Table 3. Normalization.

Cell Measurement

Table 4. Raw data of OD600 measurement.

Table 5. Blank subtraction and correction of OD600 measurement.

Figure 3. Blank subtraction and correction of Abs600 measurement.

Table 6. Raw data of fluorescence measurement.

Table 7. Blank substraction and correction of fluorescence measurement.

Figure 4. Blank subtraction and correction of fluorescence measurement.

Table 8.Raw data of Fl/Abs600.

Table 9. Raw data of average

Table 10. Raw data of SD

Figure 5. Average level of devices.

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 <div class="title" ><strong style="color:#05BAC3">Materials</strong></div>
 <br><br>
-<div class="subtitle" style="color:#05BAC3" ><strong>Plasmids used</strong></div>
 <div class="p">
+<strong style="color:#05BAC3">Plasmids used</strong>
 <ul style="list-style:none">
 <li>Positive Control Device: I20270 in pSB1C3 (Located in Kit Plate 6, well 20B)</li>
@@ Line 55: / Line 56: @@
 </ul>
 </div>
 <br><br>
-<div class="subtitle" style="color:#05BAC3" ><strong>Strain used</strong></div>
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+<strong style="color:#05BAC3">Plasmids used</strong>
 <ul style="list-style:none">
 <li>Escherichia coli strain DH5α</li>
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 </div>
 <br><br>
-<div class="subtitle" style="color:#05BAC3" ><strong>Machines</strong></div>
 <div class="p" >
+<strong style="color:#05BAC3">Plasmids used</strong>
 <ul style="list-style:none">
 <li>Thermo Multiskan FC</li>
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 <br><br>
-<div class="subtitle" ><strong style="color:#05BAC3">Sequencing</strong></div>
 <div class="p">
+<strong style="color:#05BAC3">Sequencing</strong>
 <ul style="list-style:none;margin-left:0">
 <li>Positive Control ：BBa_I20270</li>
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 <br><br>
-<div class="subtitle" ><strong style="color:#05BAC3">>BBa_I20270 Part-only sequence (919 bp)</strong></div>
 <div class="p">
+<strong style="color:#05BAC3">>BBa_I20270 Part-only sequence (919 bp)</strong>
 <p style="textindent:0;">
 ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtccc