Team:SDU CHINA/interlab

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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)

Strain used

• Escherichia coli​ strain DH5α
• Materials
• 1ml LUDOX
• H​2​0
• 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

Machines

• Thermo Multiskan FC
• BERTHOLD TECHNOLOGIES LB940Mith

Protocol

• Calibration Protocols
• Cell measurement 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.