Difference between revisions of "Part:BBa K174015"
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<partinfo>BBa_K174015 short</partinfo>
 
<partinfo>BBa_K174015 short</partinfo>
  
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This cadmium sensor is built with binding sites for two metal sensor proteins, ArsR and CzrA (1,2). They both work as repressors and they are relieved from binding to the DNA when they are bound to various metals. ArsR can bind to cadmium, silver, copper and arsenic whereas CzrA can bind to zinc, cobalt, nickel and cadmium. Cadmium can bind to both proteins and using a combinatorial approach enables us to filter out sensing other metals rather than cadmium.
+
This part is designed to allow ''B. subtilis'' to sense cadmium.
  
−
When cadmium is present both proteins are unbound from this AND gate and the promoter region controlled by the repressors becomes free to drive the expression of downstream genes such as Gfp as an indicator or other transcription factors.
+
To our knowledge, there is no specific cadmium sensing protein. We therefore combined two repressors that each sense a range of metals, but with only cadmuim in common. This cadmium sensor is therefore based on an AND gate configuration.  
  
−
To create our device we combined ArsR binding site (BBa_K174014) with ''cadA'' promoter (BBa_K174017) from ''B. subtilis''. CadA works as an efflux system to take the metals out in the cell. It has a binding site for CzrA just after the promoter region.
+
The part was built by combining operator binding sites for two metal sensor repressor proteins, ArsR and CzrA [1,2].
 +
 
 +
They both work as repressors and they are relieved from binding to the DNA when they are bound to various metals.
 +
ArsR can bind to cadmium, silver, copper and arsenic whereas CzrA can bind to zinc, cobalt, nickel and cadmium. Cadmium can bind to both proteins and using a combinatorial approach enables us to filter out sensing any metals other than cadmium.
 +
 
 +
When cadmium is present both proteins are unbound from this AND gate and the promoter region controlled by the repressors becomes free to drive the expression of downstream genes such as Gfp as an indicator, or other transcription factors.
 +
 
 +
To create our device we combined ArsR binding site (BBa_K174016) with ''cadA'' promoter (BBa_K174017) from ''B. subtilis''. CadA works as an efflux system to take the metals out in the cell. The ''cadA'' promoter was used since it has binding site for CzrA just after the promoter region. The ArsR binding site was positioned just upstream of the ''cadA'' promoter.
 
   
 
   
 
For more information about this part, go to Newcastle iGEM 2009 [http://2009.igem.org/Team:Newcastle/Project/Overview Overview] and [http://2009.igem.org/Team:Newcastle/Metalsensing Metal Sensing] pages.
 
For more information about this part, go to Newcastle iGEM 2009 [http://2009.igem.org/Team:Newcastle/Project/Overview Overview] and [http://2009.igem.org/Team:Newcastle/Metalsensing Metal Sensing] pages.
  
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<!-- Add more about the biology of this part here
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Usage and Biology
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===Usage and Biology===
+
===Characterization by Gaston Day School 2019===
 +
<p>When combined with an RBS (B0036), RFP (E1010), and a double terminator (B0014), the Gaston Day School 2019 team has shown that this promoter is sensitive to cadmium down to 0.25M (p<0.01). Work by earlier Gaston Day School teams had shown that the promoter did work, but the data was not clean and the minimum level of detection was significantly higher (approximately 10mM). Cultures were grown for 24 hours in the presence of 0, 0.25, or 0.75 mM cadmium. Absorbance was measured using a Vernier SpectroVis Plus and data was collected and analyzed using Microsoft Excel’s t-test function. Results are reported as absorbance at 517 nm normalized using absorbance at 700 nm.
 +
<!-- -->
 +
<center>https://2019.igem.org/wiki/images/a/a2/T--Gaston_Day_School--Absorbance_at_517nm_per_OD700.png</center>
  
 
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<partinfo>BBa_K174015 parameters</partinfo>
 
<partinfo>BBa_K174015 parameters</partinfo>
 
<!-- -->
 
<!-- -->
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 +
===Safety===
 +
Cadmium is a toxic metal. There are regulations and restrictions to work with heavy metals. To test this part these regulations should be followed. For more information please read [http://www.hse.gov.uk/pubns/indg391.pdf HSE's guidance] to work with cadmium.
  
 
===References===
 
===References===
−
#Moore, C. M. and J. D. Helmann (2005). "Metal ion homeostasis in Bacillus subtilis." Current Opinion in Microbiology 8(2): 188-195.
+
#Moore, C. M. and J. D. Helmann (2005). "Metal ion homeostasis in'' Bacillus subtilis''." Current Opinion in Microbiology 8(2): 188-195.
 
#Harvie, D.R., et al., Predicting metals sensed by ArsR-SmtB repressors: Allosteric interference by a non-effector metal. Molecular Microbiology, 2006. 59(4): p. 1341-1356.
 
#Harvie, D.R., et al., Predicting metals sensed by ArsR-SmtB repressors: Allosteric interference by a non-effector metal. Molecular Microbiology, 2006. 59(4): p. 1341-1356.
 +
 +
==Improved by 2022 iGEM Team Fujian_United==
 +
Our composite part BBa_K4288005 was improved based on the existing part BBa_K174015 submitted by iGEM09_Newcastle. In 2019, group iGEM_Gaston Day School combined this part with an RBS (B0036), RFP (E1010), and a double terminator (B0014), and has shown that this promoter is sensitive to cadmium down to 0.25M (p<0.01). Today, our team further improved the cadmium sensor by optimizing the sequence of cad promoter, fusing a new signal peptide of RpmG which helps protein secret outside the cell, and combining a report protein feruloyl esterase(FAE). Therefore, the new composite part BBa_K4288005 was developed.
 +
 +
 +
[[File:T-fujian-united-k4288005-figure 1.jpg|500px|thumb|center|Figure 1. The DNA sequence difference between BBa_K4288005 and BBa_ K174015).]]
 +
 +
In order to prove the function of our new composite part cadA promoter-signal peptide of RpmG-feruloyl esterase, we transformed the part in Bacillus subtilis, induced FAE protein expression with various concentrations of cadmium nitrate, and detected the enzyme activity of FAE. The result shows that our biosensor can detect the concentration of cadmium ions within 50μg/L, which broadens the concentration range of biological detection of cadmium ions and takes a more important step towards practical application.
 +
 +
=== BBa_K4288005===
 +
Name: cadA promoter-signal peptide of RpmG-feruloyl esterase
 +
 +
Base Pairs: 1128 bp
 +
 +
Origin: Bacillus subtilis, genome
 +
 +
Properties: response to the cadmium concentration
 +
====Usage and biology====
 +
The biosensor used to monitor the cadmium and zinc in the environment.
 +
====Construct design====
 +
The reporter feruloyl esterase gene controlled under the cad promoter which monitors the cadmium and zine concentration of solution. Further, the reporter fused the signal peptide RpmG. The gene fragment was inserted into the pHY300PLK vector.
 +
===BBa_K4288000===
 +
Name: cadA promoter
 +
 +
Base Pairs: 159 bp
 +
 +
Origin: Bacillus subtilis, 50S ribosomal protein
 +
 +
Properties: signal peptide
 +
====Usage and biology====
 +
The cadmium resistance in bacteria using Cad A system. in high concentration of cadmium, the cad A system uptake the cadmium by ATP-dependent pump.
 +
===BBa_K4288002===
 +
Name: signal peptide of RpmG
 +
 +
Base Pairs: 87 bp
 +
 +
Origin: Bacillus subtilis, 50S ribosomal protein
 +
 +
Properties: signal peptide
 +
====Usage and biology====
 +
BBa_K4288002 is a coding sequence of RpmG, which is an amino acid sequence of 1-29. we used it as signal peptide.
 +
===BBa_K4288001===
 +
Name: Feruloyl esterase
 +
 +
Base Pairs: 870 bp
 +
 +
Origin: E.coli
 +
 +
Properties: hydrolysis the ester bands
 +
====Usage and biology====
 +
Feruloyl esterase (FAE) produced from bacteria, fungi and plant. The enzyme mainly functions as hydrolysis the ester bands. based on amino acids sequence alignment and substrate specificities, the feruloyl esterase classified into four types, including A, B, C, and D. The type D of feruloyl esterase acts as xylanase, which is the hemicellulolytic enzyme. thus, the feruloyl esterase was used as additive to improve the bioconversion and utilization of the agricultural stuff.
 +
 +
===Introduction===
 +
As more and more human activities create accumulating impact, and as industries develop at an unprecedentedly rapid speed, huge amounts of heavy metal pollutants are released into the environment, contaminating the earth. Heavy metal ions such as Cadmium (Cd), Chromium (Cr), Lead (Pb), Mercury (Hg), and metalloids like Arsenic (As) cannot be degraded by organisms. As a result, these ions can accumulate, create chronic toxicity, and manifest bioaccumulation up the trophic level.
 +
 +
Humans then take them in without notice, either by directly drinking contaminated water or through eating other plants and animals that are previously exposed to heavy metals. If the heavy metals enriched in human bodies, they can cause serious symptoms such as diseases in the kidney, the lung, the liver, the bone, different types of cancers, and deficiency in embryonic neurodevelopment.
 +
 +
Therefore, it is urgent to develop a fast and convenient tool for the detection of heavy metals. Our project designed a cadmium biosensor to detect the heavy metal cadmium.
 +
 +
===Construction of Cadmium-biosensor plasmid===
 +
We utilize the operon CadA to expresses reporter feruloyl esterase in Bacillus subtilis and the plasmid pHY300PLK-PcadA-Biosensor was handed over to GenScript for synthesis. The synthesis report is as follows.
 +
 +
[[File:T-fujian-united-k4288005-file1.pdf|500px|thumb|center|.]]
 +
 +
===Functional test===
 +
We tested the ability of cadmium-biosensor for detecting the heavy metal cadmium. The plasmid pHY300PLK-PcadA-Biosensor was transformed into Bacillus subtilis(Fig2.) and tested while in different concentration of cadmium nitrate. The enzyme activity of feruloyl esterase(FAE) is to reflect the cadmium concentration after the induction of cadmium ions. Under different cadmium concentration scales, the enzyme activity of FAE is shown in the figure 3.
 +
 +
[[File:T-fujian-united-k4288005-figure 2.jpg|500px|thumb|center|Figure 2. Plate of the transformants of pHY300PLK-PcadA-Biosensor/Bacillus subtilis.]]
 +
 +
[[File:T-fujian-united-k4288005-figure 3.jpg|500px|thumb|center|Figure 3. The activity of Ferulyol Esterase in different concentaiton of Cd. The data were collected in 0.5h.]]
 +
 +
As shown in the graph, the more the cadmium is, the more enzyme activity it has. While, when the concentration of cadmium reaches 50μg/L, its enzyme activity value tends to be stable at about 0.35. We speculate that when the cadmium ion concentration exceeds 50μg/L, the host Bacillus subtilis cannot express the protein normally, so the measured enzyme activity value is flat. This indicates that our current cadmium biosensor is suitable for the detection of samples with cadmium ion concentration in the range of 0-50μg/L.

Latest revision as of 14:46, 12 October 2022

Cadmium Sensor

This part is designed to allow B. subtilis to sense cadmium.

To our knowledge, there is no specific cadmium sensing protein. We therefore combined two repressors that each sense a range of metals, but with only cadmuim in common. This cadmium sensor is therefore based on an AND gate configuration.

The part was built by combining operator binding sites for two metal sensor repressor proteins, ArsR and CzrA [1,2].

They both work as repressors and they are relieved from binding to the DNA when they are bound to various metals. ArsR can bind to cadmium, silver, copper and arsenic whereas CzrA can bind to zinc, cobalt, nickel and cadmium. Cadmium can bind to both proteins and using a combinatorial approach enables us to filter out sensing any metals other than cadmium.

When cadmium is present both proteins are unbound from this AND gate and the promoter region controlled by the repressors becomes free to drive the expression of downstream genes such as Gfp as an indicator, or other transcription factors.

To create our device we combined ArsR binding site (BBa_K174016) with cadA promoter (BBa_K174017) from B. subtilis. CadA works as an efflux system to take the metals out in the cell. The cadA promoter was used since it has binding site for CzrA just after the promoter region. The ArsR binding site was positioned just upstream of the cadA promoter.

For more information about this part, go to Newcastle iGEM 2009 [http://2009.igem.org/Team:Newcastle/Project/Overview Overview] and [http://2009.igem.org/Team:Newcastle/Metalsensing Metal Sensing] pages.

Usage and Biology

Characterization by Gaston Day School 2019

When combined with an RBS (B0036), RFP (E1010), and a double terminator (B0014), the Gaston Day School 2019 team has shown that this promoter is sensitive to cadmium down to 0.25M (p<0.01). Work by earlier Gaston Day School teams had shown that the promoter did work, but the data was not clean and the minimum level of detection was significantly higher (approximately 10mM). Cultures were grown for 24 hours in the presence of 0, 0.25, or 0.75 mM cadmium. Absorbance was measured using a Vernier SpectroVis Plus and data was collected and analyzed using Microsoft Excel’s t-test function. Results are reported as absorbance at 517 nm normalized using absorbance at 700 nm.

T--Gaston_Day_School--Absorbance_at_517nm_per_OD700.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 200
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Safety

Cadmium is a toxic metal. There are regulations and restrictions to work with heavy metals. To test this part these regulations should be followed. For more information please read [http://www.hse.gov.uk/pubns/indg391.pdf HSE's guidance] to work with cadmium.

References

  1. Moore, C. M. and J. D. Helmann (2005). "Metal ion homeostasis in Bacillus subtilis." Current Opinion in Microbiology 8(2): 188-195.
  2. Harvie, D.R., et al., Predicting metals sensed by ArsR-SmtB repressors: Allosteric interference by a non-effector metal. Molecular Microbiology, 2006. 59(4): p. 1341-1356.

Improved by 2022 iGEM Team Fujian_United

Our composite part BBa_K4288005 was improved based on the existing part BBa_K174015 submitted by iGEM09_Newcastle. In 2019, group iGEM_Gaston Day School combined this part with an RBS (B0036), RFP (E1010), and a double terminator (B0014), and has shown that this promoter is sensitive to cadmium down to 0.25M (p<0.01). Today, our team further improved the cadmium sensor by optimizing the sequence of cad promoter, fusing a new signal peptide of RpmG which helps protein secret outside the cell, and combining a report protein feruloyl esterase(FAE). Therefore, the new composite part BBa_K4288005 was developed.


Figure 1. The DNA sequence difference between BBa_K4288005 and BBa_ K174015).

In order to prove the function of our new composite part cadA promoter-signal peptide of RpmG-feruloyl esterase, we transformed the part in Bacillus subtilis, induced FAE protein expression with various concentrations of cadmium nitrate, and detected the enzyme activity of FAE. The result shows that our biosensor can detect the concentration of cadmium ions within 50μg/L, which broadens the concentration range of biological detection of cadmium ions and takes a more important step towards practical application.

BBa_K4288005

Name: cadA promoter-signal peptide of RpmG-feruloyl esterase

Base Pairs: 1128 bp

Origin: Bacillus subtilis, genome

Properties: response to the cadmium concentration

Usage and biology

The biosensor used to monitor the cadmium and zinc in the environment.

Construct design

The reporter feruloyl esterase gene controlled under the cad promoter which monitors the cadmium and zine concentration of solution. Further, the reporter fused the signal peptide RpmG. The gene fragment was inserted into the pHY300PLK vector.

BBa_K4288000

Name: cadA promoter

Base Pairs: 159 bp

Origin: Bacillus subtilis, 50S ribosomal protein

Properties: signal peptide

Usage and biology

The cadmium resistance in bacteria using Cad A system. in high concentration of cadmium, the cad A system uptake the cadmium by ATP-dependent pump.

BBa_K4288002

Name: signal peptide of RpmG

Base Pairs: 87 bp

Origin: Bacillus subtilis, 50S ribosomal protein

Properties: signal peptide

Usage and biology

BBa_K4288002 is a coding sequence of RpmG, which is an amino acid sequence of 1-29. we used it as signal peptide.

BBa_K4288001

Name: Feruloyl esterase

Base Pairs: 870 bp

Origin: E.coli

Properties: hydrolysis the ester bands

Usage and biology

Feruloyl esterase (FAE) produced from bacteria, fungi and plant. The enzyme mainly functions as hydrolysis the ester bands. based on amino acids sequence alignment and substrate specificities, the feruloyl esterase classified into four types, including A, B, C, and D. The type D of feruloyl esterase acts as xylanase, which is the hemicellulolytic enzyme. thus, the feruloyl esterase was used as additive to improve the bioconversion and utilization of the agricultural stuff.

Introduction

As more and more human activities create accumulating impact, and as industries develop at an unprecedentedly rapid speed, huge amounts of heavy metal pollutants are released into the environment, contaminating the earth. Heavy metal ions such as Cadmium (Cd), Chromium (Cr), Lead (Pb), Mercury (Hg), and metalloids like Arsenic (As) cannot be degraded by organisms. As a result, these ions can accumulate, create chronic toxicity, and manifest bioaccumulation up the trophic level.

Humans then take them in without notice, either by directly drinking contaminated water or through eating other plants and animals that are previously exposed to heavy metals. If the heavy metals enriched in human bodies, they can cause serious symptoms such as diseases in the kidney, the lung, the liver, the bone, different types of cancers, and deficiency in embryonic neurodevelopment.

Therefore, it is urgent to develop a fast and convenient tool for the detection of heavy metals. Our project designed a cadmium biosensor to detect the heavy metal cadmium.

Construction of Cadmium-biosensor plasmid

We utilize the operon CadA to expresses reporter feruloyl esterase in Bacillus subtilis and the plasmid pHY300PLK-PcadA-Biosensor was handed over to GenScript for synthesis. The synthesis report is as follows.

File:T-fujian-united-k4288005-file1.pdf

Functional test

We tested the ability of cadmium-biosensor for detecting the heavy metal cadmium. The plasmid pHY300PLK-PcadA-Biosensor was transformed into Bacillus subtilis(Fig2.) and tested while in different concentration of cadmium nitrate. The enzyme activity of feruloyl esterase(FAE) is to reflect the cadmium concentration after the induction of cadmium ions. Under different cadmium concentration scales, the enzyme activity of FAE is shown in the figure 3.

Figure 2. Plate of the transformants of pHY300PLK-PcadA-Biosensor/Bacillus subtilis.
Figure 3. The activity of Ferulyol Esterase in different concentaiton of Cd. The data were collected in 0.5h.
As shown in the graph, the more the cadmium is, the more enzyme activity it has. While, when the concentration of cadmium reaches 50μg/L, its enzyme activity value tends to be stable at about 0.35. We speculate that when the cadmium ion concentration exceeds 50μg/L, the host Bacillus subtilis cannot express the protein normally, so the measured enzyme activity value is flat. This indicates that our current cadmium biosensor is suitable for the detection of samples with cadmium ion concentration in the range of 0-50μg/L.