Difference between revisions of "Part:BBa I20260:Experience"

iGEM12_OUC-China

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).

iGEM14_Paris_Bettencourt

iGEM Paris Bettencourt Team 2014 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 BioBrick registry as BBa_K1403000. Thus, the devices are exactly the same contruct: constitutive Anderson's promoter J23115 and a GFP generator. The only difference was the vector.

We could observe a remarkable difference in GFP expression efficiency

Mean OD600 absorbance measured over 20h. Background absorbance (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for each sample).

Mean of green fluorescence for BBa_I20260, BBa_K1403000 and NEB turbo cells. Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates).

Mean of green fluorescence divided by optical density 600 for BBa_I20260 and BBa_K1403000 and NEB turbo cells. Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates)

UNIQa0254bf9fc0bee30-partinfo-00000005-QINU

Conlusions

In the OD600 vs. time graphs we can see that the growth rate of BBa_I20260 and BBa_K1403000 are comparabe. The BBa_K1403000 shows better efficiency of GFP expression. It is an expected result at it is placed in a high copy pSB1C3 plasmid, while BBa_I20260 is placed in a low copy plasmid pSB1K3.

We can also read the general GFP expression efficiency under J23101 Anderson's constitutive promoter when comparing with the NEB turbo control without any fluorescent vector (expression only natural cell fluorescence)

Protocol

We followed iGEM Distribution Kit instructions to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1# Heat Shock transformation of E.coli]. For successful Chloramphenicol plates, form single colonies we prepared liquid cultures overnight. We used 750uL of the liquid cultures for a [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8# glycerol stock] . We used remaining 4,25 mL to make [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4# minipreps]. We measured DNA content with the nanodrop.

Digestion analysis:
- 5 ug plasmid
- 5 ul FD Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)

We made an eletrophoresis gel to check the fragments (the bands at around 876 bp for GFP and 2100 bp for the promoter + backbone) and then extract BBa_E0240 with Gel extraction kit. For the plasmid with the promoter we used a [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot5# PCR purification kit]. We introduced the GFP fragment to the promoter + backbone through ligation of the sticky ends SpeI and XbeI. Quantified DNA in two parts with nanodrop. The amount of vector: insert has been calculated with Promega calculator.

5X Ligase Reaction Buffer 4 μl
Insert: Vector Molar Ratio 1:1, 1:3, 1:5
Total DNA 0.01-0.1 μg
T4 DNA Ligase 1 uL
Autoclaved distilled water to 25uL
Incubate at 22°C for 1h
16°C overnight

We transformed the ligation product following [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1# Heat Shock transformation of E.coli]. We have put a single colony into a liquid culture with the appropriate antibiotic and the next day we prepared a [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8# glycerol stock]

Measurment

Samples preparation
Single colonies were inoculated in 5mL LB broth with appropriate antibiotic and grown to saturation overnight (16h) at 37°C with shaking (220 rpm). Samples were diluted 100x (50um in 5 mL LB with appropriate antibiotic) and incubated for 2h at 37°C prior to measurement.

Control
LB broth with antibiotics (chloramphenicol/kanamycin)- no fluorescence.
NEB turbo without fluorescence - no fluorescence, no cells.

Measurment
Greiner 96 plates were loaded with 150um of cells in LB and 30um mineral oil Cells have been diluted prior to measurement as described above. Background absorbance and fluorescence was determined from LB control.
Data from the top row were excluded due to the likely evaporation and artefacts (edge effects).

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.

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]