Part:BBa_I20260:Experience

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User Reviews

UNIQb0f5e9489380e542-partinfo-00000000-QINU UNIQb0f5e9489380e542-partinfo-00000001-QINU

No review score entered. Paris_Bettencourt 2014

We participated in the Interlab study 2014. We constructed a device BBa_J23101 + BBa_E0240 (B0032-E0040-B0015) in sPB1C3 which BBa_I20260 in sPB1C3 and we submitted it to the BioBric registry as BBa_K1403000. To compare the promoter activity we also submitted BBa_K1403001 which BBa_K823012 (mutated J23115 + BBa_E0240 (B0032-E0040-B0015).

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••••• iGEM12_OUC-China

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We improved this part,and submit the improved partBBa_K737068 to the parts registry. The absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. Researchers should choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. Drew Endy, etc demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by ~50%. They defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. [1] However, unfortunately, there are few data about it and few teams use it as an in vivo control. This year, we detected many RFU (Relative Fluorescence Unit) curves and made it as control group. Our team wants to upload some data about it and hope other teams can use it to decrease the error. These are RFU curves of K737069, Top10 and I20206 (in PSB4A5). These are RFU curves of K737052 and I20206 (in PSB4A5). Reference [1] Kelly, J.r. et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. J. Biol. Eng. 3, 4 (2009).

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UNIQb0f5e9489380e542-partinfo-00000005-QINU

Characterization

••••• iGEM2014 [http://2014.igem.org/Team:XMU-China# XMU-China]

iGEM2014 [http://2014.igem.org/Team:XMU-China# XMU-China] has sequenced this device. The following data was taken from this device.

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We choose the promoter J23101 as an in vivo reference standard for promoter activity. In order to get the different expression intensity of BioBrick backbones, we can connect the backbones with the same promoter and GFP generator. Finally we can use the intensity of fluorescent protein as a charaterization data to report the expression intensity of different backbones. Unfortunately, there are few data about it. So we detected the two different backbones as examples and hope the other teams can provide more relevant data. For devices BBa_I20260 and BBa_J23101 + BBa_E0240. Both devices consist of Anderson promoter J23101 and GFP generator BBa_E0240. When the device is constructed in backbone pSB3K3. A low copy number is in expectation, as a result, a weak fluorescence strength is shown. While the device is constructed in pSB1C3 which is a higher copy number vector, hence a stronger fluorescence strength, so that it can be obvious enough to be observed in naked eyes. So we reconstructed this part, and submit the improved part BBa_K1412716 and BBa_K1412924 to the parts registry. BBa_K1412716: BBa_I20260 (J23101-B0032-E0040-B0015), in the pSB3K3 vector. BBa_K1412924: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), in the pSB1C3 vector. Measurenment Figure 1. The plot of optical density versus time From the plot of optical density versus time, we can conclude that the growth rate of bacteria is become lower with time. We measured the samples three times parallelly, and we can know that the reproducibility of the data is acceptable. When we compare BBa_K1412716 with BBa_K1412924, we can get that their growth rate are almost equal. Figure 2. The plot of RFUs versus time From the plot of RFUs versus time, we can conclude that RFUs grows linearly with time. When we compare BBa_K1412716 with BBa_K1412924, we can get that the fluoresent expression intensity of BBa_K1412924 is higher than BBa_K141716, because the pSB1C3 backbone has a higher copy number, hence we can get a stronger fluorescence strength. Figure 3. The plot of RFUs versus OD600 From the plot of RFUs versus OD600, we can conclude that RFUs grow linearly with OD600 both BBa_K1412716 and BBa_K1412924. Because the fluoresent protein expression of each bateria is contain, so when the concentration of bacteria increase, the fluoresent expression increase too. When we compare BBa_K1412716 with BBa_K1412924, we can get that the fluoresent expression intensity of BBa_K1412716 is lower than BBa_K1412924. Figure 4. The plot of RFUs/OD600 versus time From the plot of RFUs/OD600 versus time, we know the RFUs/OD600 is a representation of the fluoresent expression intensity of unit bacteria. So we can get that the fluoresent expression intensity of BBa_K1412716 is lower than BBa_K1412924. Protocol 1. Transformed BBa_K1412924 into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃. 2. Inoculate a 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin or Kanamycin 50 μg/ml) with single colony from the plate. 3. Cultures were grown in conical flask for 16 hrs at 37℃ with shaking at 200 rpm in the table concentrator. 4. Cultures were diluted 1:100 into three 20 ml fresh LB medium and grown for 3 hrs at 37℃ with shaking at 200 rpm in the table concentrator. 5. Then transfered 650 μl of the culture to a 1.5 ml centrifuge tube, centrifuged and washed twice with phosphate-buffered saline ([http://en.wikipedia.org/wiki/Phosphate_buffered_saline PBS], pH 7.4) to minimize the background fluorescence from the medium. 6. The washed cells were suspended in [http://en.wikipedia.org/wiki/Phosphate_buffered_saline PBS] and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements. 7. Measure the fluorescence and absorbance: (1)Fluorescence: Device: [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers SpectraMax+M5 microplate reader], 96-well plates. Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm. (2)OD600 (optical density at 600 nm): Device: [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers SpectraMax+M5 microplate reader], 96-well plates. Wavelengths: 600 nm absorption. 8. Measure every 30 minutes in the next 4 hrs. References [1] [http://journals.aps.org/pre/abstract/10.1103/PhysRevE.82.021911 Bagh, Sangram, Mahuya Mandal, and David R. McMillen. "Minimal genetic device with multiple tunable functions." Physical Review E 82.2 (2010): 021911] More information, click here: [http://2014.igem.org/Team:XMU-China# XMU-China]