Difference between revisions of "Part:BBa I0500:Experience"

Antiquity

This review comes from the old result system and indicates that this part worked in some test.

iGEM Groningen 2009

The sequence was listed as inconsistent, and the ligations of parts behind the promoter failed. Restriction of isolated plasmids showed fragments of unexpected sizes.

[http://2010.igem.org/Team:Slovenia 2010 iGEM team Slovenia]

[http://2010.igem.org/Team:Slovenia 2010 iGEM team Slovenia] further characterized the pBAD/araC promoter (Part:Bba_I0500) using lacZ gene that encodes for beta-galactosidaze enzyme (Part:Bba_K323133). pBAD promoter is an arabinose inducible promoter. When incubating E.coli cultures containing the lacZ under pBAD/araC promoter, increasing concentrations of arabinose leads to higer promoter activity that results in higher amounts and subsequently higer activity of beta-galactosidase enzyme as seen from figure below. The resulting graph shows a trend of increasing beta-galactosidase activity over increasing concentrations of arabinose.

Beta-galactosidase activity of cultures with different concentrations of arabinose increases with raising the arabinose concentration.

The tested cultures incubated at different concentrations of arabinose (i.e. 0%, 0.001%, 0.0025%, 0.005%, 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75% and 1%) have shown a trend of increasing beta-galactosidase activity with raising arabinose concentration. The activity was highest at 1% added arabinose, which implies the promotor should be induced by this arabinose concentration for reaching maximum expression of the regulated protein. However, some beta-galactosidase activity was observed even at 0% added arabinose, which indicates slight leakage of the pBAD promoter.

pBAD/araC_lacZ_DTER construct (Part:Bba_K323133) includes a beta-galactosidase (lacZ) gene, the expression of which is regulated by the pBAD promoter (Part:Bba_I0500). This construct was designed for the sole purpose of characterising the pBAD promoter with the beta-galactosidase assay (for a detailed description of the method see the 2010 iGEM team Slovenia wiki).

[http://2011.igem.org/Team:Groningen 2011 iGEM team Groningen]

[http://2011.igem.org/Team:Groningen 2011 iGEM team Groningen] characterized the pBAD/AraC promoter (Part:Bba_I0500) even further using GFP and measuring the fluorescence in a fluorimeter over time, utilizing different arabinose concentrations to induce the promoter. After analysis of the obtained data (see graphs below), it is clear that the induction is faster and stronger with higher arabinose concentrations. It shows a dynamic response over a range of arabinose concentrations and over time. Because of that it can be treated as a precise tool capable of giving different output levels.

The measurements were done in DH5alpha strain carrying a pSB1C3 with BBa_K607036 in triplicate.

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  Figure 1a. Cells carrying a pSB1C3 plasmid with BBa_K607036, induced at time=0h.

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  Figure 1b. Wild type cells, induced at time=0h.

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  Figure 2a. Cells carrying a pSB1C3 plasmid with BBa_K607036, induced at time=0h.

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  Figure 2b. Wild type cells, induced at time=0h.

[http://2011.igem.org/Team:Cambridge Cambridge 2011]

We observed an 'all-or-nothing' response in our use of the pBAD promoter. We grew up cells of the strain [http://cgsc.biology.yale.edu/Strain.php?ID=111773 BW27783] overnight at 37°C. We then diluted the culture 10-fold and grew the cells back up to an OD_600 of 0.5 to catch the cells in mid-exponential phase. We then induced with arabinose at various concentrations from 0.001mM up to 10mM and took OD and GFP readings on a plate reader over the next 20 hours. During this time the cells were held at 37&degrees;C. The cells contained our ReflectinA1-sfGFP and TorA-ReflectinA1-sfGFP constructs on pSB3K3 plasmids.

This work was not intended to characterise the pBAD promoter, but it highlights some of the difficulties in doing so:

• In contrast to the Groningen team we observed an 'all-or-nothing' response, with very little fluorescence at [ara] of OM or 0.001mM and high fluorescence readings at 0.01mM or above, with little difference between the readings.
• [http://www.ncbi.nlm.nih.gov/pubmed/11739756 This paper] reports that response to arabinose is highly variable between different strains of E coli.
• Induction also altered the growth pattern of our cells. The fluorescence readings need to be corrected not just for OD but also for cell state.

Note: 1mM arabinose corresponds to 0.015% w/v.

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  center | 600px | OD readings of cells carrying K638301 on a pSB3K3 backbone, after induction with arabinose at time 0h.

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  center | 600px | GFP fluorescence readings of cells carrying K638301 on a pSB3K3 backbone, after induction with arabinose at time 0h.

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  center | 600px | OD readings of cells carrying K638403 on a pSB3K3 backbone, after induction with arabinose at time 0h.

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  center | 600px | GFP fluorescence readings of cells carrying K638403 on a pSB3K3 backbone, after induction with arabinose at time 0h.

[http://2012.igem.org/Team:UNITN-Trento Trento 2012]

We were unable to extract this part from the distribution kit, so we developed our own! Please head to BBa_K731201 for documentation and characterization.

[http://2014.igem.org/Team: Hong_Kong_HKUST 2014]

2014 HKUST iGEM team previously submitted [http://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization characterization data] of Part BBa_I0500 that described an all-or-none behavior of the promoter. That is however not the case. We sincerely apologize for the misleading information. Please refer to our latest characterization data by HKUST-Rice 2015 below instead.

UNIQc5b440c9c66d06d9-partinfo-0000000A-QINU

[http://2015.igem.org/Team: HKUST-Rice 2015]

[http://2015.igem.org/Team:HKUST-Rice HKUST-Rice Team] characterized BBa_I0500 using the construct BBa_I13540 in low copy plasmid pSB3K3. We aimed to obtain a dose response curve capturing the dynamic range and maximum induction concentration in order to compare its behavior when it is [http://2015.igem.org/Team:HKUST-Rice/Expression expressed in parallel with another sensor]. (please see BBa_K1682017 for more details.

Figure 1. Dose response curve of cells carrying pSB3K3-BBa_I13540 under increasing L-arabinose concentration. Fluo = Relative Fluorescence Units. Error bars represent SD (n=3). Results indicate that the sensing range of BBa_I0500 lies at roughly 10^-3 to 10^-1 mM and has a half-maximal induction concentration of around 3^-2mM of Arabinose.

Characterization was done using E. coli strain DH10B. Overnight inoculated cell cultures in M9 supplemented medium with corresponding arabinose concentrations were diluted 20 fold in medium with arabinose the next day. Measurements for population-averaged assay using fluorometry were taken after 5.5 hours of induction and incubation at 37°C when the OD600 was around 0.3-0.4.

We have also fixed characterization data from last year for the construct BBa_I2031 on low copy plasmid pSB3K3 and high copy plasmid pSB1K3. In BBa_I2031, BBa_I0500 is driving expression of a reporter GFP in which translation is initiated by medium RBS BBa_B0032).

Our results demonstrated that this promoter has a graded induction response and different sensing ranges on plasmids with different copy numbers (Figure 2). In concordance with literature, it displays an all-or-none behavior on a single cell level when expressed from a high copy plasmid pSB1K3, but not when expressed from a low copy plasmid pSB3K3 (Figure 3).

Figure 2. Transfer curves for BBa_I2031 on plasmid pSB3K3 and pSB1K3. On pSB3K3, BBa_I0500 is responsive to 10^-4 - 10^-2 mM arabinose, whereas on pSB1K3, it senses arabinose from roughly 10^-3 to 1mM. These data agreed with neither Cambridge 2011 nor Groningen 2011’s result. The dashed lines represent cells’ auto fluorescence, measured using DH10B cells harboring pSB3K3-BBa_E0240 or pSB1K3-BBa_E0240. Error bars represent SEM of 3 independent experiments on 3 different days. The molar concentration to percentage conversion was provided for comparison with previous data.

Figure 3. Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid. An all-or-none induction can be observed when BBa_I0500 is placed on a high copy plasmid, where induced cells were mostly distributed among two bins of fluorescence. Yet on low copy plasmid, the induced populations remain homogenous along the arabinose concentration gradient. Concentrations of arabinose for high copy pSB1K3 plasmid: 0.488µM – 0.25mM. For low copy pSB3K3 plasmid: 0.0610µM – 0.03125mM. Only 1 set of experiment result from 3 replicates is presented.

Moreover, we would like to warn potential users of this part not to drive expression of riboregulators ([http://www.ncbi.nlm.nih.gov/pubmed/15208640 Isaacs et al., 2004]) using BBa_I0500. This P_BAD promoter contains a 19bp sequence after the transcription start site, which would produce a taRNA with additional nucleotides at its 5’ end, and rendering it incapable to activate crRNA. To avoid misuse of this part due to incognizance of its sequence information, we recommend users to look up BBa_K1067007 for annotations. For more information and methods of our characterization, please refer to our detailed characterization report available through our [http://2015.igem.org/Team:HKUST-Rice/Expression/ParaBAD wiki page].

[http://2016.igem.org/Team:IISc_Bangalore]Team IISc Bangalore

Fluorescence versus time curve for sfGFP expression under pBAD/araC promoter

IISc Bangalore characterized BBa_I0500 by using Bba_746908 which has sfGFP under the pBAD/araC promoter. We wanted to verify the catabolite repression of the expression from the promoter in the presence of glucose.
E. coli BL21 (DE3) was transformed with BBa_746908 and a single colony was inoculated into LB broth (with the appropriate concentration of antibiotic) primary that was grown overnight (37C, 180rpm) and then 1% of the primary was inoculated into 8 x 250mL conical flasks, 2 each with the following:
1. 100mL LB broth (blank)
2. 100mL LB broth + 0.2% glucose
3. 100mL LB broth + 0.2% arabinose
4. 100mL LB broth + 0.2% arabinose + 0.2% glucose
0.5mL samples were collected every hour in microfuge tubes and stored at 4C and the flasks were incubated at 37C, 180rpm. OD600 and fluorescence values of the samples were recorded. The data was plotted and the plots are shown.
The fluorescence data indicated the trend expected, the fluorescence at almost all times showed the following trend:
Glucose < Glucose + Arabinose < Blank << Arabinose
This is the expected trend as arabinose induces expression from the promoter in the absence of glucose (CAP site occupied by CAP-cAMP complex) and in the presence of glucose (independent of the availability of arabinose), the expression from the promoter is greatly repressed due to the low concentration of cAMP inside the cell, however, the expression in glucose + arabinose will be slightly higher than just glucose as there will be more sites occupied by CAP-cAMP complex in glucose + arabinose than in glucose. In the blank (LB broth) very low concentrations of glucose and arabinose are present and the expression from the promoter will very low. The above trend is exactly in accordance with the information available in the literature about pBAD/araC promoter, thus, we verified for the biobrick Bba_I0500 that catabolite repression is seen, glucose is a repressor and also indirectly verified the presence of a CAP site in the biobrick. We are OUC-China. Previously, we found that the discription of the part is concentrated upon the level of protein expression. In order to verify the reliability and rigour of the part and find the best induced concentration, we decide to explore the strength of promoter that different induced concentration of arabinose may result in from the level of RNA and protein. Here are our data. 1. 100mlLB，blank 2. 100mlLB,0.0002%arobinose 3. 100mlLB,0.002%arobinose

4. 100mlLB,0.02%arobinose

5. 100mlLB,0.2%arobinose <img src=""/> <img src=""/> <img src=""/> According to our data analysis, when induced with 0.02% arabinose, our strain TOP10 was effected better than that induced with 0.2% according to the previous research. We infer that 0.2% arabinose may affect the growth of our strain. So, in the future, we suggest others to use this concentration to induce the TOP10.