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.

UNIQ5d1144dcdab60b49-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.

[http://2016.igem.org/Team:OUC-China]Team OUC-China

[http://2016.igem.org/Team:OUC-China OUC-China] characterized BBa_I0500 of different concentrations of L-arabinose on the transcriptional level. We found that the strength of I0500 was mainly measured on the translational level before. As far as we are concerned, it is the transcriptional level that can directly reflect I0500’s original strength without interfering by context. In order to verify it more accurately, we tested it on the transcriptional level by extracting RNA and measured it by qPCR. We constructed I0500 with GFP(E0040) and transformed into TOP10F’. Firstly , we plated on LB agar with chloramphenicol and individual colonies were subsequently grown overnight in LB-medium. Cultures were back diluted 1:100 into 100mL fresh LB-medium with chloramphenicol, adding 0,0.0002%,0.002%,0.02%,0.2% (m/V)L-arabinose. After growing for 18 hours, we extracted RNA and measured it. At the same time, we measured the fluorescence of GFP(485nm/520nm). Besides, we also measured OD over time to check whether L-arabinose promotes bacteria’s growth.

Figure1 OD after induction with different concentration of L-arabinose over time

Figure2 The relative expression level of GFP after induction with different concentration of L-arabinose.

1. 100mlLB，blank
2. 100mlLB,0.0002%arobinose
3. 100mlLB,0.002%arobinose
4. 100mlLB,0.02%arobinose
5. 100mlLB,0.2%arobinose
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.

[http://2017.igem.org/Team:Glasgow]Team Glasgow 2017

[http://2017.igem.org/Team:Glasgow Team Glasgow 2017] improved this part by splitting its constituent parts into two separate BioBricks: BBa_K2442101 encoding minimal pBAD and BBa_K2442104 encoding AraC under regulation of LacI-regulated promoter. This allows for greater control over arabinose-inducible systems. Placing AraC under control of LacI regulated promoter (R0011) allows to induce expression of AraC only when required, taking translational load off grown cells. R0011 is inducible by IPTG. AraC coding sequence alone is also available as part BBa_K2442103, which can be placed under other promoter of choice.

We isolated the minimal DNA sequence from the native pBAD that is sufficient to retain its function. The promoter retains all the operator sites O₁, O₂, I₁ and I₂ required for binding of AraC. As some of these sites lie within the PC promoter and overlap with AraC coding sequence, the minimal pBAD has the araC start codon ATG mutated to AGT to prevent the protein from being made. See part BBa_K2442101 and our [http://2017.igem.org/Team:Glasgow/araC wiki] for details.

We tested pBAD with the use of pBAD+GFP reporter plasmid (BBa_K2442102) by measuring fluorescence. Activity of the promoter was tested in presence of arabinose, xylose, decanal and no inducer (Fig. 1). When transformed into AraC-positive E. coli strains such as DH5α, pBAD is sufficiently activated with just the chromosomal copy of araC and does not require introduction of araC from external plasmid. Moreover, DH5α cells carrying K2442102 showed fluorescence in presence of arabinose, but no significant increase in fluorescence in other conditions, showing high specificity of AraC and minimal leakage of minimal pBAD. DH5α carrying pBAD+GFP reporter on a high copy number plasmid (pSB1C3) BBa_K2442102 showed 2000x fold increase in fluorescence on arabinose plate compared to empty cells (Fig. 1). This proves significant improvement of part BBa_I0500. DH5α cells carrying minimal pBAD and I13500 on a low copy number plasmid pSB3K3 instead of pSB1C3 also showed expression of GFP only under arabinose conditions. However, levels of fluorescence were much lower, suggesting that the level of GFP production is dependent on the plasmid copy number and must be taken into account in further experiments. In AraC-negative strains, AraC expressed from BBa_K2442104 induces pBAD with similar efficiency.

Overall, results show that both AraC and arabinose are necessary to activate pBAD. Efficiency of minimal pBAD is demonstrated by 2000x fold increase in fluorescence of cells carrying the reporter plasmid.

Figure 1:Average relative fluorescence over optical density at hour 8. Shows fluorescence levels under: no additive, 40mM Arabinose, 40mM Xylose and 2mM decanal. The E. coli strains which the plasmids have been transformed into are DS941 or DH5α (specified). DH5α is AraC-positive. DS941 is AraC-negative. BioBricks (K244210..) specified. All parts are in plasmid backbone pSB1C3 unless specified otherwise (pSB3k3). K2442102: reporter plasmid pBADmin+GFP. K2442104: regulatory plasmid R0011+B0032+AraC

BHSF_ND iGEM 2019

We mainly focused on two types of inducible transcriptional promoter—Xyls and pBAD. The Xyls protein is the positive transcription regulator of the TOL plasmid meta-cleavage pathway operon Pm. Xyls belongs to the AraC family of transcriptional regulators and exhibits an N-terminal domain involved in effector recognition, and a C-terminal domain.The activator Xyls is usually is usually activated by the benzoate and its derivatives (3MBz in our design). The pBAD promoter holds great potential to control the expression of genes that require tight regulation, as it is capable of both an induction and repression. The pBAD promoter exhibits an almost all-or-none behavior upon induction with arabinose when located on a high copy vector, but allows for gradual induction when cloned into a low copy vector. Based on these research findings, a low copy vector was used to investigate the ability to inhibit gene expression subsequent to induction of pBAD.

As the result shows, both system exhibit relative high level of fluorescence compared to our backbone design( BN006/Contains BBa_K3202029) which suggest the two inducible transcription system function properly.

Figure 1: pBAD(BN007/Contains BBa_K3202030) induction curve at different concentrations after 4 hours compared to backbone.(BN006/Contains BBa_K3202029)

Figure 2: Xyls(BN009/Contains BBa_K3202031) induction curve at different concentrations after 4 hours compared to backbone(BN006/Contains BBa_K3202029)

Figure3: Time induction curve pBAD(BN007/Contains BBa_K3202030) at the concentration of 1mM compared to backbone (BN006/Contains BBa_K3202029)

Figure4: Time induction curve for Xyls(BN009/Contains BBa_K3202031) at the concentration of 1mM compared to backbone (BN006/Contains BBa_K3202029)

Results

In our experiment of leakage testing, we used low-copying plasmid backbone PSB4K5, with a PSC101 origin of replication.

Two inducers with their respective promoters (AraC & pBAD; Xyls & Pm) are coupled with sfGFP to see if there is actually expression leakage when inducer is present quantitatively through flow cytometry. We tested fluorescence under different inducer concentration at the given time of 240 minutes and under different time stage at a given inducer concentration. Theoretically when inducers (AraC and 3MBz ) are present, promoters (pBAD and Pm) are initiated therefore both sfGFP are expressed; while sfGFP shouldn’t be expressed if inducer is absent. When measured at the end of 240 minutes without inducer, BN009/Contains BBa_K3202031(Xyls+sfGFP) has a basal leakage of 968 a.u. compared to BN006/Contains BBa_K3202029(backbone without GFP). BN007/Contains BBa_K3202030(pBAD+sfGFP) also showed a leakage of 90 a.u. The result suggested that both system have noticeable leakage and Xyls has much higher leakage.

When we gradually increased inducer concentration to 1mM, the fluorescence of BN006/Contains BBa_K3202029 stays at a constant level, whereas the fluorescence of both Xyls and pBAD have increased exponentially. This is another sign that both system function properly.

We found an intriguing result when measuring the fluorescence of the two system under a constant inducer concentration of 1mM. During the first 160 minutes, the fluorescence of BN007/Contains BBa_K3202030 is 200 a.u. higher than BN006/Contains BBa_K3202029. However, at 240 minutes, the fluorescence value suddenly increased to 7641 a.u., which is 7590 a.u., higher than the backbone. Same thing happened to BN009/Contains BBa_K3202031. Therefore, in the subsequent experiments, systems are induced to up to 4 hours to ensure the accuracy of the experiment. Results shown above verified the presence of expression leakage of both systems when inducer is not expressed through the detectable fluorescence of reporter protein using flow cytometry.