iGEM Lund 2019

Results and Discussion

This is the page were we present our results. This is where we discover if genetically modified gut bacteria can be a part of the solution against exposure to toxic metals.

Overview

We have successfully sequenced the following parts:

BBa_K3282003 (arsR-T7)
BBa_K3282004 (arsR-Tac)

We have successfully transformed E.coli Nissle 1917 to absorb:

~40% of lead in a concentration of ~2 mg/L, in 2.5 hours.
~20% of arsenic in a concentration of ~16 mg/L, in 2.5 hours.

We have failed on the following points:

To see evidence of protein expression using SDS-page.
To sequence the parts BBa_K3282005 (pbrD-T7) and BBa_K3282007 (pbrD-Tac).

Parts

The following parts were designed:

Composite part with arsR and a T7 promoter: BBa_K3282003

Composite part with arsR and a Tac promoter: BBa_K3282004

Composite part with pbrT, pbrD and a T7 promoter: BBa_K3282005

Composite part with pbrT, pbrD and a Tac promoter: BBa_K3282007

From now on the parts will be reffered to as the following:

arsR-T7: BBa_K3282003
arsR-Tac: BBa_K3282004
pbrD-T7: BBa_K3282005
pbrD-Tac: BBa_K3282007

Isolation of E. coli Nissle 1917

E. coli Nissle 1917 (EcN) was isolated from a probiotic pharmaceutical Mutaflor. It was resuspended and plated on MacConkey agar that is used to select several E. coli strains, among them EcN [1]. In order to confirm the identity of the strain a colony PCR was performed with primers specific for genomic island II (GEI II) and for cryptic plasmid pMUT2 that are specific for EcN. For GEI II the bands were expected at 400bp, while for pMUT2 at 313 bp [2,3]. The results of the colony PCR can be seen in figure 1 below.

Figure 1: Colony PCR result showing well-defined bands with various annealing temperatures, and pMUT2 and GEI II primers.

All colonies picked from MacConkey agar have shown the expected band with GEI II primers but no bands at all with pMUT2. These results confirm the identity of the strain to be EcN, despite negative results for pMUT2 primers since the GEI II primers are specific for chomrosomal region of EcN [2].

Sequencing of Composite Parts

The following parts were sequenced ars-T7, ars-Tac, pbrD-T7 and pbrD-Tac.

The sequences of ars-T7 and ars-Tac showed 100% and 99.6% similarity, the alignment results can be found here and here respectively. For ars-Tac there are two base substitutions in the sequence. The first one is in the region that is only used for amplification PCR and hence it had no effect on protein structure or expression. However, the second substitution is in the protein coding region and resulted in R18L mutation.

Unfortunately, in the case of pbrD-T7 and pbrD-Tac, samples were mixed up or contaminated with ars-T7 and ars-Tac and therefore it was not possible to align the sequences. Due to lack of time it was not possible to repeat sequencing for those composite parts.

Cultivation of Transformed Bacteria

The E. coli BL21(DE3) strain was transformed with biobrick ars-T7 in pUC19 plasmid by heat shock. Similarly, the BL21(DE3) was transformed with part pbrD-T7 and EcN strain with part pbrD-Tac in pUC19. Unfortunately, arsR-Tac arrived late and cultivation could therefore not be performed. The growth rate of the transformed and wild type strains were compared by cultivating the cells in LB media at 37 °C and measuring OD every hour. The cultivation was carried out to investigate if the presence of plasmid acts as a burden for the cells. As is evident from figure 2, the growth rate of the control and the transformed bacteria are very similar. This is unexpected since arsR expression has shown to be toxic to cells [4]. This conclusion is a positive indicator that the cells would not die before their intended use of absorbing arsenic.

Figure 2: Comparison of the growth rate of E. coli BL21(DE3) transformed with ars-T7 inserted into pUC19 and wild type E. coli BL21(DE3).

Figure 3: Comparison of growth rate of E coli BL21(DE3) transformed with pbrD-T7 inserted into pUC19 and wild type E coli BL21(DE3).

The growth rate of E. coli BL21(DE3) containing the lead construct was compared with the wild type BL21(DE3). Figure 3 displays growth of cells over time. The growth rate of the cells containing the construct had a growth rate of 0.474 h^-1, and the control of BL21(DE3) cells had a growth rate of 0.263 h^-1. The cells containing the constructs grow at a pace that is a bit faster than the control. This result is unexpected, since cells that are forced to express heterologous proteins often have a lower growth rate because of the burden it bears on the cell. The linear fit is also not perfect, with low R² values compared to what would be wanted. The results could be accidental. However, the results also give an indicator that the cells would not die before they have a chance to perform the desired function of lead uptake.

Figure 4: Comparison of growth rate of EcN transformed with pbrD-Tac inserted into pUC19 and wild type EcN.

Figure 4 above shows the growth rate of both transformed and untransformed EcN containing pUC19 with the lead construct. The results are analogous to the E. coli BL21(DE3). The growth rate of the EcN containing the plasmid and the control are similar, with the cells containing the construct exhibiting a slightly higher pace. Growth rates of transformed EcN was 0.697 h^-1, while the rate for the untransformed control was 0.556 h^-1. Since the result is similar to the results in figure 3, so is the discussion. The results could be random from looking at the relatively poor linear fit. The plasmid does from this result not appear to be a burden to the cell and would have a chance to perform the desired function of lead uptake before cell death.

Toxic Metal Analysis

Investigation of Composite Parts for Arsenic Accumulation

The part ars-T7 consists of a T7 promoter (BBa_I719005), Anderson RBS (BBa_J61109), arsenic resistance operon repressor (BBa_K3282000) and TE terminator (BBa_B0012), whereas ars-Tac (arsR-Tac)comprises the same basic parts but with a Tac promoter (BBa_K3282006).

The Escherichia coli chromosomal ars operon confers resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony. ArsR is the first gene in the ars operon containing a very specific binding site towards As(III) [4].

Experiment

ars-T7 was inserted into pUC19 and successfully transformed in E. coli BL21(DE3), and ars-Tac was transformed in the probiotic strain EcN. arsR protein expression was demonstrated by performing SDS-PAGE. The cells were cultured in enriched media (containing peptone, yeast extract, phosphates, chlorides and sulphates) at 37°C. They were grown overnight and were harvested in their late exponential phase in order 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 wild type E. coli BL21(DE3) and wild type EcN in growth medium containing 50 µM As₂O₃.

Results

The SDS-PAGE analysis result can be seen in figure 5 and figure 6. The expected band for arsR protein has a molecular weight of 13.5 kDa, therefore it should appear between the two marked points as shown in figure 5 and 6. The gel shows that the engineered bacterial strains, as well as the negative controls have a band with the expected molecular weight. Thus, we can not 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 5: Samples were harvested at different time points, and the total cellular proteins were analyzed by SDS-PAGE. Protein bands of BL21(DE3) can be seen after induction with IPTG, showing arsR at 13.5 kDa. The control shown is wild type BL21(DE3) without arsR.

Figure 6: Samples were harvested at different time points, and the total cellular proteins were analyzed by SDS-PAGE. Image with protein bands of EcN after induction with IPTG, showing ArsR at 13.5 kDa. The control shown is wild EcN without arsR.

Arsenic accumulation over time

Figure 7: Arsenic concentration in media contaning BL21 transformed with the composite part ars-T7 or non-transformed E. coli BL21 (DE3).

Arsenic accumulation over time at varying pH

Figure 8: Arsenic accumulation over time at varying pH. The blue line represents the arsenite concentration in a culture maintained at pH 3 containing the composite part while the red line represents arsenite concentration in a culture maintained at pH 5 containing the composite part. The yellow line represents the arsenite concentration in a culture with wild type EcN.

Figure 7 and figure 8 compare the arsenite accumulation over time whereas figure 9 and figure 10 compare the growth of engineered as well as wild type strains. It can be seen in figure 7 that the initially added arsenite had a concentration of 3.745 mg/L for both engineered as well as wild type BL21(DE3), and the arsenite concentration measured at 0 hours was 0.014 mg/L and 0.013 mg/L respectively. Similarly, it can be seen in figure 8 that the initially added arsenite had a concentration of 3.745 mg/L (ars-Tac at pH3 and Control) and 14.98 mg/L (ars-Tac at pH5), and the arsenite concentration measured at 0 hours was 0.012 mg/L, 0.012 mg/L, and 0.024 mg/L respectively. This rapid decrease in both cases was unexpected. The relative concentration of arsenic in media where transformed and non transformed bacteria were present is showcased in figure 11.

Growth comparison between transformed and non-transformed E. coli BL21(DE3)

Figure 9: Comparison of growth of engineered BL21(DE3) with wild type BL21(DE3) in the presence of arsenic oxide.

Growth comparison between transformed and non-transformed EcN

Figure 10: Comparison of growth of engineered EcN with wild type EcN in the presence of arsenic oxide.

Relative Concentration of Arsenite in Media Containing Transformed and Non-Transformed Bacteria

Figure 11: Relative concentration of arsenite in growth media at different timepoints (0, 2.5 and 5 h) with transformed and untransformed (control) strains of E. coli.

Discussion

Arsenite does not suffer from sorption to glass surfaces as it forms oxy ions which are partly dissociated leading to negatively charged ions [5]. Therefore, this decrease cannot be accounted for by the sticking of ions to the glass surface. 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 [7]. 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, plasmid lacking 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 [6]. The wild type E. coli strains are resistant to arsenite up to a concentration of 1 mM. This might explain the similarity in growth and arsenite uptake between the engineered EcN and BL21(DE3) strains, and the wild type strains. However, if the measured arsenite concentration at 0 hour is compared with the concentration at 2.5 hours, the engineered BL21(DE3) shows a 21 % accumulation of arsenite, compared with only 8 % arsenite accumulation in the control. Similarly, for EcN, the comparison of measured arsenite concentration at 0 hours with the concentration at 2.5 hours, shows that the engineered EcN accumulated 22 % arsenite, whereas the control accumulated 13 % arsenite. This can be considered as a proof of concept; showing that the engineered EcN and BL21(DE3) strains express arsR and have an improved ability to uptake arsenite. The slight increase in the arsenite concentration at 5 hours might suggest that some cells died and released the accumulated arsenite. The growth curves in figure 10 and figure 11 imply that the wild type, as well as engineered strains were able to survive in the presence of arsenite and at pH 5. However, EcN had a very low growth rate at pH 3, suggesting that the cumulative effect of low pH and the presence of toxic arsenite might be inhibitory for the cells.

Investigation of Composite Parts for Lead Accumulation

pbrD-T7 composite part consists of a T7 promoter (BBa_I719005), two Anderson RBS (Part:BBa_J61109), separating two coding regions of lead binding protein (BBa_K3282002) and lead transport protein (BBa_K3282001), followed by a TE terminator (Part:BBa_B0012), whereas the pbrD-Tac (pbrD-Tac) comprises the same basic parts but with a Tac promoter (BBa_K3282006).

The pbrD gene encodes a Pb(II)-binding protein which is essential for functional lead sequestration, whereas the expression of pbrT leads to uptake of lead into the cytoplasm to reduce interaction of free Pb(II) with side chains of membrane and periplasmic proteins, which would cause extensive cellular damage [8].