Difference between revisions of "Part:BBa K3282004"

Latest revision as of 14:01, 20 October 2019

ArsR under Tac promoter

The part consists of Tac promoter (Part:BBa_K3282006), Anderson RBS (Part:BBa_J61109), arsenic resistance operon repressor (Part:BBa_K3282000) and TE terminator (Part:BBa_B0012).

The Escherichia coli chromosomal ars operon confers resistance to arsenicals by a specific efflux pump controlled by an arsenite-inducible repressor, arsR which has been shown to be a trans-acting repressor that senses environmental As(III)[1].

Experiment

This part was ligated into pUC19 and transformed into E. coli Nissle. The expression of arsR was demonstrated by performing Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. The cells were cultured in enriched media (containing peptone, yeast extract, phosphates, chlorides and sulphates) at 37 °C. They were grown overnight followed by cell harvesting to normalize the OD values to 2. After normalization, the cells were resuspended in fresh enriched medium containing 10 µM, 50 µM and 200 µM of As2O3. As a negative control, the experiment was also performed with the non engineered E. coli Nissle in growth medium containing 50 µM As2O3. Samples were collected every hour for OD measurement. For investigating the arsenic accumulation by engineered bacteria, samples were collected at 0 hour, 2.5 hour and 5 hour after the addition of As2O3.

Results

The SDS-PAGE analysis result can be seen in Figure 1. arsR has a molecular weight of 13.5 kDa, which is expected to be between the two bands marked in Figure 1. The gel shows that the engineered bacteria as well as the control have a band with the expected molecular weight. Thus, we cannot confirm that it corresponds to arsR. It is possible that there are other proteins expressed in E. coli of the same molecular weight, masking the expression of arsR.

Figure 1: Image with protein bands of E. coli Nissle after induction with IPTG, showing arsR at 13.5 kDa. Samples were harvested at different time points, and the total cellular proteins were analyzed by SDS-PAGE. The control shown is non-engineered E. coli Nissle without arsR.

Figure 2: Comparison of arsenite concentration in the growth medium containing E. coli Nissle cells expressing arsR and E. coli Nissle cells without arsR expression.

Figure 3: Comparison of growth rate of engineered E. coli Nissle with wild type E. coli Nissle.

The Figure 2 compares the arsenite accumulation over time whereas Figure 3 compares the growth rate of engineered as well as non-engineered EcN 1917. It can be seen that the initial added arsenite had the concentration of 3.745mg/L (K3282004 at pH3 and Control) and 14.98mg/L (K3282004 at pH5) and the arsenite concentration measured at 0 hour was 0.012 mg/L, 0.012 mg/L and 0.024 mg/L respectively. This rapid decrease in the concentration was not expected. This rapid decrease cannot be accounted for by the sticking of ions to the glass surface since it has been shown that arsenite does not suffer from sorption to the glass surfaces as it forms oxy ions which is partly dissociated leading to negatively charged ions [2].It might be the case that the engineered bacteria as well as the control have a capacity to uptake arsenite. According to Chen et al., when the cells are depleted of endogenous energy reserves, arsenite enters whether or not the cells carry a resistance plasmid [3]. The ars operon carried on the Escherichia coli R factor R773 encodes the transport system that extrudes arsenite and the lowering of the intracellular concentration of toxic oxyanion produces resistance, however, plasmidless strains of E. coli have also been shown to be intrinsically resistant to arsenite due to the presence of chromosomal ars operon. It has also been found that the R773 arsR protein is 75% identical with the chromosomal product in E. coli strains [4]. The wild type E. coli strains are resistant to arsenite upto the concentration of 1 mM. This might explain the similarity in growth and arsenite uptake between the engineered strain and the wild type strain. However, if the measured arsenite 0 hour concentration is compared with the final concentration at 5 hour, the engineered EcN shows a 21.83 % arsenite accumulation compared with only 12.89 % arsenite accumulation in the control. This can be considered as a proof of concept showing that the engineered bacteria express arsR and has an improved ability to uptake arsenite.

From the Figure 3, it can be seen that both the control and the engineered strain with arsR were able to survive in an adverse environment of pH 5 and the presence of As2O3. However, the growth rate is much less at pH 3. This can mean that the cells cannot survive well under pH 3 because of the cumulative effect of low pH and toxic As2O3.

Future Improvements

Since there was no visible protein expression at the expected position, one could try to add a histidine tag, making purification of protein possible and one would be able to assess the protein expression. Another solution would be to use a stronger promoter, which would give a higher expression of protein, and might be visible on the SDS-PAGE. Another improvement could be to make sure the protein is not stuck in the wells of the gel. According to Kostal et al. (2004) overexpression of arsR can be toxic to the host. It was found that fusing arsR with elastin polypeptide or maltose binding protein can reduce the toxic effect and hence improve the overexpression of arsenic accumulation protein.

References

1. Kostal, J., Yang, R., Wu, C. H., Mulchandani, A., & Chen, W. (2004). Enhanced arsenic accumulation in engineered bacterial cells expressing ArsR. Applied and environmental microbiology, 70(8), 4582–4587. doi:10.1128/AEM.70.8.4582-4587.2004.

2. Massee, R., Maessen, F. (1981). Losses of silver, arsenic, cadmium, selenium, and zinc traces from distilled water and artificial sea-water by sorption on various container surface. Analytica Chimica Acta, 127, pp. 206-210.

3. Chen, C., Misra, T., Silver, S., Rosen, B. Nucleotide sequence of the structural genes for an anion pump: the plasmid-encoded arsenical resistance operon. (1986). J. Biol. Chem., 261, pp. 15030-15038.

4. Carlin, A., Shi, W., Dey, S., & Rosen, B. P. (1995). The ars operon of Escherichia coli confers arsenical and antimonial resistance. Journal of bacteriology, 177(4), 981–986. doi:10.1128/jb.177.4.981-986.1995.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 198
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 The ''Escherichia coli'' chromosomal ''ars'' operon confers resistance to arsenicals by a specific efflux pump controlled by an arsenite-inducible repressor, ''arsR'' which has been shown to be a trans-acting repressor that senses environmental As(III)[1].
 '''Experiment'''
-This part was ligated into pUC19 and transformed into ''E. coli'' Nissle. The expression of ''arsR'' was demonstrated by performing SDS-PAGE analysis. The cells were cultured in enriched media (containing peptone, yeast extract, phosphates, chlorides and sulphates) at 37 °C. They were grown overnight followed by cell harvesting to normalize the OD values to 2. After normalization, the cells were resuspended in fresh enriched medium containing 10 µM, 50 µM and 200 µM of As2O3. As a negative control, the experiment was also performed with the non engineered ''E. coli'' Nissle in growth medium containing 50 µM As2O3.
+This part was ligated into pUC19 and transformed into ''E. coli'' Nissle. The expression of ''arsR'' was demonstrated by performing Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. The cells were cultured in enriched media (containing peptone, yeast extract, phosphates, chlorides and sulphates) at 37 °C. They were grown overnight followed by cell harvesting to normalize the OD values to 2. After normalization, the cells were resuspended in fresh enriched medium containing 10 µM, 50 µM and 200 µM of As2O3. As a negative control, the experiment was also performed with the non engineered ''E. coli'' Nissle in growth medium containing 50 µM As2O3.
 Samples were collected every hour for OD measurement. For investigating the arsenic accumulation by engineered bacteria, samples were collected at 0 hour, 2.5 hour and 5 hour after the addition of As2O3.
 '''Results'''
 The SDS-PAGE analysis result can be seen in '''Figure 1'''. ''arsR'' has a molecular weight of 13.5 kDa, which is expected to be between the two bands marked in '''Figure 1'''. The gel shows that the engineered bacteria as well as the control have a band with the expected molecular weight. Thus, we cannot confirm that it corresponds to ''arsR''. It is possible that there are other proteins expressed in E. coli of the same molecular weight, masking the expression of ''arsR''.
 [[File:K3282004-SDS.png]]
-'''Figure 1 Bioaccumulation of arsenite.Samples were harvested at different time points, and the total cellular proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Image with protein bands of ''E. coli'' Nissle after induction with IPTG, showing arsR at 13.5 kDa. The control shown is non-engineered ''E. coli'' Nissle without ''arsR''.'''
+'''Figure 1: Image with protein bands of ''E. coli'' Nissle after induction with IPTG, showing ''arsR'' at 13.5 kDa. Samples were harvested at different time points, and the total cellular proteins were analyzed by SDS-PAGE. The control shown is non-engineered ''E. coli'' Nissle without ''arsR''.'''
 [[File:K3282004-TM.png]]
-'''Figure 2 : Comparison of arsenite concentration in the growth medium containing ''E. coli'' Nissle cells expressing ''arsR'' and ''E. coli'' Nissle cells without ''arsR'' expression.'''
+'''Figure 2: Comparison of arsenite concentration in the growth medium containing ''E. coli'' Nissle cells expressing ''arsR'' and ''E. coli'' Nissle cells without ''arsR'' expression.'''
 [[File:K3282004-OD.png]]
-'''Figure 3 Comparison of growth rate of engineered ''E. coli'' Nissle with non-engineered control of ''E. coli'' Nissle.'''
+'''Figure 3: Comparison of growth rate of engineered ''E. coli'' Nissle with wild type ''E. coli'' Nissle.'''
-The '''Figure 2''' compares the arsenite accumulation over time whereas '''Figure 3''' compares the growth rate of engineered as well as non-engineered EcN 1917. There are no significant differences between the K3282004 and the control in both arsenite accumulation levels and growth. The initial concentration in the flask was 3.745mg/L (K3282004 at pH3 and Control) and 14.98mg/L (K3282004 at pH5) and at time 0h the measured concentrations are  0.012mg/L and 0.024mg/L. This shows that 99.87 % arsenite was accumulated by the engineered EcN strain at pH 5 compared with 99.68 % arsenite accumulation by the control. The reduction in arsenite concentration cannot be accounted for anything else other than accumulation since it has been shown that arsenic does not suffer from sorption to the glass surfaces as it forms oxy ions which is partly dissociated leading to negatively charged ions [2]. The ''ars'' operon carried on the ''Escherichia coli'' R factor R773 encodes the transport system that extrudes arsenite and the lowering of the intracellular concentration of toxic oxyanion produces resistance, however, plasmidless strains of ''E. coli'' have also been shown to be intrinsically resistant to arsenite due to the presence of chromosomal ''ars'' operon [3]. It has also been found that the R773 ''arsR'' protein is 75% identical with the chromosomal product in ''E. coli'' strains. The wild type ''E. coli'' strains are resistant to arsenite upto the concentration of 1 mM. This might explain the similarity in growth and arsenite uptake between the engineered strain and the wild type strain [3]. According to Chen et al., when the cells are depleted of endogenous energy reserves, arsenite enters whether or not the cells carry a resistance plasmid [4]. So to conclude, it might be the case that the ''arsR'' protein is not expressed in the cells since it is toxic for the cells, but they survived and accumulated arsenite due to chromosomal ''ars'' operon.
-From the '''Figure 3''', it can be seen that both the control and the engineered strain with ''arsR'' were able to survive in an adverse environment of pH 5 and the presence of As2O3. However, the growth rate is much less at pH 3. This can mean that the cells cannot survive well under pH 3 because of the adverse conditions.
+The '''Figure 2''' compares the arsenite accumulation over time whereas '''Figure 3''' compares the growth rate of engineered as well as non-engineered EcN 1917. It can be seen that the initial added arsenite had the concentration of 3.745mg/L (K3282004 at pH3 and Control) and 14.98mg/L (K3282004 at pH5) and the arsenite concentration measured at 0 hour was 0.012 mg/L, 0.012 mg/L and 0.024 mg/L respectively. This rapid decrease in the concentration was not expected. This rapid decrease cannot be accounted for by the sticking of ions to the glass surface since it has been shown that arsenite does not suffer from sorption to the glass surfaces as it forms oxy ions which is partly dissociated leading to negatively charged ions [2].It might be the case that the engineered bacteria as well as the control have a capacity to uptake arsenite. According to Chen et al., when the cells are depleted of endogenous energy reserves, arsenite enters whether or not the cells carry a resistance plasmid [3]. The ''ars'' operon carried on the ''Escherichia coli'' R factor R773 encodes the transport system that extrudes arsenite and the lowering of the intracellular concentration of toxic oxyanion produces resistance, however, plasmidless strains of ''E. coli'' have also been shown to be intrinsically resistant to arsenite due to the presence of chromosomal ''ars'' operon. It has also been found that the R773 ''arsR'' protein is 75% identical with the chromosomal product in ''E. coli'' strains [4]. The wild type ''E. coli'' strains are resistant to arsenite upto the concentration of 1 mM. This might explain the similarity in growth and arsenite uptake between the engineered strain and the wild type strain. However, if the measured arsenite 0 hour concentration is compared with the final concentration at 5 hour, the engineered EcN shows a 21.83 %  arsenite accumulation compared with only 12.89 % arsenite accumulation in the control. This can be considered as a proof of concept showing that the engineered bacteria express ''arsR'' and has an improved ability to uptake arsenite.
+From the '''Figure 3''', it can be seen that both the control and the engineered strain with ''arsR'' were able to survive in an adverse environment of pH 5 and the presence of As2O3. However, the growth rate is much less at pH 3. This can mean that the cells cannot survive well under pH 3 because of the cumulative effect of low pH and toxic As2O3.
@@ Line 38: / Line 42: @@
 Since there was no visible protein expression at the expected position, one could try to add a histidine tag, making purification of protein possible and one would be able to assess the protein expression. Another solution would be to use a stronger promoter, which would give a higher expression of protein, and might be visible on the SDS-PAGE. Another improvement could be to make sure the protein is not stuck in the wells of the gel. According to Kostal et al. (2004) overexpression of ''arsR'' can be toxic to the host. It was found that fusing ''arsR'' with elastin polypeptide or maltose binding protein can reduce the toxic effect and hence improve the overexpression of arsenic accumulation protein.
 '''References'''
 . Kostal, J., Yang, R., Wu, C. H., Mulchandani, A., & Chen, W. (2004). Enhanced arsenic accumulation in engineered bacterial cells expressing ArsR. Applied and environmental microbiology, 70(8), 4582–4587. doi:10.1128/AEM.70.8.4582-4587.2004.
 . Massee, R., Maessen, F. (1981). Losses of silver, arsenic, cadmium, selenium, and zinc traces from distilled water and artificial sea-water by sorption on various container surface. Analytica Chimica Acta, 127, pp. 206-210.
-. Carlin, A., Shi, W., Dey, S., & Rosen, B. P. (1995). The ars operon of Escherichia coli confers arsenical and antimonial resistance. Journal of bacteriology, 177(4), 981–986. doi:10.1128/jb.177.4.981-986.1995.
-. Chen, C., Misra, T., Silver, S., Rosen, B. Nucleotide sequence of the structural genes for an anion pump: the plasmid-encoded arsenical resistance operon. (1986). J. Biol. Chem., 261, pp. 15030-15038.
+. Chen, C., Misra, T., Silver, S., Rosen, B. Nucleotide sequence of the structural genes for an anion pump: the plasmid-encoded arsenical resistance operon. (1986). J. Biol. Chem., 261, pp. 15030-15038.
+. Carlin, A., Shi, W., Dey, S., & Rosen, B. P. (1995). The ars operon of Escherichia coli confers arsenical and antimonial resistance. Journal of bacteriology, 177(4), 981–986. doi:10.1128/jb.177.4.981-986.1995.
 <!-- Add more about the biology of this part here