Part:BBa_K3282005
pbrD and pbrT under T7 promoter
The part consists of 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).
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 [1].
Cultivation of Transformed bacteria
The E. coli BL21(DE3) strain was transformed with biobrick Pb-T7 in pUC19 plasmid by heat shock. 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 1, the growth rate of the control and the transformed bacteria are very similar.
The growth rate of E. coli BL21(DE3) containing the lead construct was compared with the wild type BL21(DE3). Figure 1 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 1: Comparison of growth rate of E coli BL21(DE3) transformed with pbrD-T7 inserted into pUC19 and wild type E coli BL21(DE3).
Toxic Metal Analysis
This part was ligated into pUC19 and transformed into E. coli BL21(DE3). The expression of pbrD and pbrT 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. The cells 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 Pb(NO3)2. As a negative control, the experiment was also performed with the non engineered E. coli BL21(DE3) in growth medium containing 50 µM Pb(NO3)2. Samples were collected every hour for OD measurement. For investigating the lead accumulation by engineered bacteria, samples were collected at 0 hour, 2.5 hour and 5 hour after the addition of Pb(NO3)2.
Results
The SDS-PAGE analysis result can be seen in Figure 2. The pbrD is a 26.7 kDa and pbrT 68.3 kDa protein. Therefore the bands were expected just above the 25 kDa band and just below 70 kDa band for pbrD and pbrT respectively. The gel shows that the engineered bacteria as well as the control have bands with the expected molecular weight. Thus, we cannot confirm that the bands corresponds to the desired proteins. It is possible that there are other proteins expressed in E. coli of the same molecular weight, masking the expression of pbrD and pbrT.
Figure 2: Image with protein bands of BL21(DE3) after induction with IPTG, showing pbrD at 26.7 kDa and pbrT at 68.3 kDa. Samples were harvested at different time points, and the total cellular proteins were analyzed by SDS-PAGE. The control shown is wild type BL21(DE3) without pbrD and pbrT.
Figure 3: Comparison of lead uptake by engineered BL21(DE3) with wild type BL21(DE3).
Figure 4: Comparison of growth rate of engineered BL21(DE3) with wild type BL21(DE3).
The initial lead concentration added to both engineered and wild type cells was 10.63 mg/L and the concentration at 0 hour was 0.63 mg/L and 0.66 mg/L respectively. This rapid decrease in the concentration was not expected.
A percentage of this rapid decrease can be attributed to the use of borosilicate Erlenmeyer flasks since it was found that borosilicate glass can adsorb lead ions [2]. However, the study also mentions that adsorption of lead by borosilicate glass is time dependent and adsorption was found to be only 17.2% over a period of 20 hours. This concludes that not all the lead adsorption was by borosilicate glass but also by the bacterial strains. If the values at time 0 hour and 2.5 hour are compared, it can be seen that the engineered EcN showed a 39.68 % lead accumulation while only 7.58 % accumulation by the control. This can be considered as a proof of concept showing that the engineered bacteria express pbrT and pbrD and has an improved ability to uptake lead. The final lead concentrations were 0.76 mg/L for engineered BL21(DE3) and 0.59 mg/L for the control. The increase in the lead concentration from 2.5 hour to 5 hour can be attributed to the fact that the cells died and released the accumulated lead. This corroborates with the growth data shown in Figure 4 where it can be seen that the engineered cells have reached stationary phase.
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. Further, fluorescent protein reporters such as GFP and RFP can be used for more convenient protein detection and stronger output signals as shown by Xiaoqiang, J et al [3].
References
1. Borremans, B., Hobman, J. L., Provoost, A., Brown, N. L., & van Der Lelie, D. (2001). Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. Journal of bacteriology, 183(19), 5651–5658. doi:10.1128/JB.183.19.5651-5658.2001
2. Kim, N. D., & Hill, S. J. (1993). Sorption of lead and thallium on borosilicate glass and polypropylene: Implications for analytical chemistry and soil science. Environ Technol, 14(11), 1015-1026. doi:10.1080/09593339309385378
3. Xiaoqiang Jia, Tingting Zhao, Yilin Liu, Rongrong Bu, Kang Wu, Gene circuit engineering to improve the performance of a whole-cell lead biosensor, FEMS Microbiology Letters, Volume 365, Issue 16, August 2018, fny157, https://doi.org/10.1093/femsle/fny157
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1444
Illegal NheI site found at 1642 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1392
Illegal NgoMIV site found at 1855 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1999
Illegal SapI.rc site found at 2229
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