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Revision as of 15:34, 18 October 2017


Tongji iGEM
TongJi iGEM
TongJi iGEM
InterLab
We partecipated in the 4th iGEM Interlab study along with about 100 teams. We have focused on quantifying the expression of GFP in common, comparable or absolute units.

Background & Design

Our team took part in this study which aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in different units. In our case, we measured fluorescence using a plate reader.
It is very common to use fluorescence as a proxy for promoter activity, and the green fluorescent protein (GFP) is the most popular protein. Although detecting the intensity of fluorescence is an indirect measurement, we can use it as representative of the expression levels of GFP. This method has a significant advantage, which is that it allows to monitor continuously without disrupting cells.
Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.
We aim to proceed in the experiment using the supplied FITC as a standard reference material. The standard curve can be constructed by measuring the fluorescence of a dilution series. We have previously performed this standard curve on our own instrument alongside a standard curve for purified GFP. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform a relative measurements of fluorescence into an absolute measurements of GFP.
However, we aim to contain instrument variability, at least to some degree, by measuring a standard scattering solution of a mono-dispersed silica suspension (LUDOX). The objective is to see if a simple, single fixed-point measurement can be used as a ratiometric adjustment to provide greater uniformity in fluorescence/OD600 measurements across sites.

Description

Plasmids containing promoters and GFP were taken from The 2017 DNA Distribution Kit 7 and all devices were transformed into E. coli.
3ml of liquid LB-M medium with chloramphenicol were inoculated with two chosen colonies of each device. Liquid cultures were incubated for 16~18 hours in HONOUR INCURATOR SHAKER placed in incubator. OD of these cultures was measured by MAPADA UV-3100PC SPECTROPHOTOMETER and diluted to 0.02. Fluorescence of biological and also technical replicates was measured using Thermo VARIOSKAN FLASH following our protocol.

Results

Sequencing
Length Sequence
BBa_R0040 Part-only sequence 54 bp tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac
BBa_J23101 Part-only sequence 35 bp tttacagctagctcagtcctaggtattatgctagc
BBa_J23106 Part-only sequence 35 bp ttacggctagctcagtcctaggtatagtgctagc
BBa_J23117 Part-only sequence 35 bp ttgacagctagctcagtcctagggattgtgctagc
BBa_J364100 Part-only sequence 84 bp gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggttttct
BBa_B0010 Part-only sequence 80 bp ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc
BBa_B0012 Part-only sequence 41 bp tcacactggctcaccttcgggtgggcctttctgcgtttata
BBa_E0040 Part-only sequence 720 bp atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtg atgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaa acttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtc actactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatg actttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaaga tgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaataga atcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaat acaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagt taacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaa caaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacac aatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgt aacagctgctgggattacacatggcatggatgaactatacaaataataa
Table 1 - OD600 Reference Point
LUDOX-HS40 H2O
Replicate 1 0.00624 -0.00726
Replicate 2 0.00684 -0.00526
Replicate 3 0.00734 -0.00366
Replicate 4 0.01104 -0.00476
Arithmetic Mean 0.007865 -0.00524
Corrected Abs600 0.0131
Reference OD600 0.0425
OD600/Abs600 3.244275
Table 2 - FITC Standard Curve
uM Fluorescein Replicate 1 Replicate 2 Replicate 3 Replicate 4 Arith. Mean Arith. Std.Dev.

50

61138.13773

65271.06373

64591.86073

65095.54373

64024.15148

1945.425461

25

46595.20773

47958.14973

47759.33273

46973.06773

47321.43948

644.4444304

12.5

28000.11673

29885.96173

29220.19473

29151.63573

29064.47723

783.0590871

6.25

15683.49573

16760.13373

16218.20373

16178.31273

16210.03648

440.0474392

3.125

8520.498733

8943.591733

8732.387733

8904.756733

8775.308733

193.0857332

1.5625

3763.249733

4130.652733

3895.511733

4038.104733

3956.879733

161.3000976

0.78125

1812.487733

2054.542733

1959.396733

2020.259733

1961.671733

106.9557819

0.390625

985.464733

1054.489733

1024.705733

1069.755733

1033.603983

37.14713908

0.1953125

497.078733

527.161733

514.866733

522.469733

515.394233

13.21958841

0.09765625

244.808733

261.900733

248.052733

257.166733

252.982233

7.919506613

0.048828125

119.568733

122.370733

123.219733

127.955733

123.278733

3.48646784

0

0.419733

0.411733

0.046733

0.241733

0.279983

0.175837757

Methods & Materials

Strain used: E. coli DH5-alpha
Plasmid DNA (100 pg/uL in 10uL of water)
Positive Control (BBa_I20270): J23151.B0032.E0040.B0010.B0012 in pSB1C3 Negative Control (BBa_R0040): R0040 in pSB1C3 Test Device 1 (BBa_J364000): J23101.B0034.E0040.B0010.B0012 in pSB1C3 Test Device 2 (BBa_J364001): J23106.B0034.E0040.B0010.B0012 in pSB1C3 Test Device 3 (BBa_J364002): J23117.B0034.E0040.B0010.B0012 in pSB1C3 Test Device 4 (BBa_J364003): J23101.J364100.E0040.B0010.B0012 in pSB1C3 Test Device 5 (BBa_J364004): J23106.J364100.E0040.B0010.B0012 in pSB1C3 Test Device 6 (BBa_J364005): J23117.J364100.E0040.B0010.B0012 in pSB1C3
Materials
FITC Standard: one tube with dried down FITC for creating a FITC standard LUDOX: one tube with 30% colloidal silica suspended in 1mL of water 1xPBS (phosphate buffered saline) LB (Luria Bertani) media Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH) 50 ml Falcon tube (or equivalent) or 250 ml shake flask for cell growth 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Pipettes Black 96 well plate
Machines: SpectraMax M5, SPX-150B-Z biochemical incubator and THZ-312 Thermostatic oscillator Software: Microsoft Excel and pro5 Calibration: OD600 Reference point and FITC fluorescence standard curve Cell measurement: Transformation and Measurements
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