Part:BBa_K4023003
glpF-rbs-modified MTIA
This is a bicistronic gene containing a glpF, a ribosome binding siteand a modified MTIA. This gene is designed to coexpress the glycerol uptake facilitator protein (coded by glpF) with the modified metallothionein IA protein. The glycerol uptake facilitator protein transports arsenite into E.coli. This could help to remove the bottleneck in As III uptake, potentially improving the efficiency of arsenite remediation1. Thus This bicistronic gene has been optimized to express the glycerol uptake facilitator protein and modified metallothionein IA protein for increased remediation of As III from the environment.
Characterization of Composite part
Characterization of composite part was performed by wetlab and drylab kinetic modelling.
Experiments and Methods
The experimental design can be found on our wiki in the Experimentstab. Due to resitrictions imposed by the COVID 19 situation, the gene was synthesized via IDT, and transformation and verification was performed by UW Biofab. We collected the successfully transformed and streaked plates of BL21 DE3 E.coli from UW Biofab and induced protein expression by inoculating a colony of transformed E.coli in MagicMedia™ E. coli Expression Medium overnight. An aliquot of the induced bacteria were lysed for protein expression, while the rest were collected for metal tolerance assay.
Protein concentration of lysate was analyzed with Nanodrop, and protein concentration was diluted to 2mg/ml with 1x Laemmli buffer. The samples were then loaded into precast SDS gel and ran for ~30min at 200V. The gel was then stained with Coomassie Blue for 2 hours and destained with destaining solution, changed every 30min accompanied by gentle agitation.
The metal tolerance assay involves the determination of the minimum inhibitory concentration of Arsenite on the successfully transformed bacteria. Briefly, initial concentration of bacteria was determined and the induced bacteria were diluted. Meanwhile LB broth containing various concentration of Sodium Arsenite solution (between 0mM to 10mM) was prepared. Subsequently, 25ul of diluted induced bacteria and 175ul of LB broth with Sodium Arsenite were added in 96 well microplates. The plates were incubated at 37 degree Celsius, and the absorbance at OD600 was taken after 20hrs of incubation.
Results
Due to the limited amount of time we had in the lab, the data we gathered are preliminary and requires further experimentations to improve reliability and accuracy. Nevertheless they are promising data and bodes well to the feasibility of our system design and of the intended function of the modified protein.
SDS PAGE
The size of MT proteins are around 6kDa whereas the size of glpF protein is approximately 28kDa. From the gel, MTs are found in all 6 lanes, indicating that the 6 cultures of bacteria containing 6 different plasmids all managed to express MTs successfully. A similar trend is observed for glpF. This is expected as while only 3 E.coli cultures contained the bicistronic gene, glpF is a major intrinsic protein in E.coli and hence would be naturally to. Thus going forward, to verify the overexpression of glpF, a western blot analysis could be performed.
Metal Tolerance
E.coli containing glpF-MTIA, glpF-4MT2 and glpF-Modified MTIA (glpF-UWMT1) all experienced a decrease in viability as concentration of arsenite increased. There is no significant difference between the data points in E.coli viability between 2-6 mM Arsenic. However at 10mM the arsenite tolerance of E.coli containing glpF-Modified MTIA (glpF-UWMT1) is significantly lower than the other 2. Given that this data is merely preliminary, more rounds of experimentations would need to be performed to verify this phenomenon.
Comparing the results between E.coli containing bicistronic glpF-MT and just the MT gene, there is little difference in the arsenite tolerance in the 2 groups. Hence it is safe to say the although co-expressing glpF will increase transport of arsenite into E.coli, it would not result in a significant rise in cell toxicity and decrease E.coli tolerance of arsenite. Hence it is a viable system to use for remediation of arsenite.
Kinetic Modeling
The kinetics modelling team set out to investigate the feasibility of regulating expression of the composite part. We modeled the kinetics of the expression of GlpF and our modified MT within E. coli. The expression of GlpF and MT in our model is controlled by the transcription factor arsR. More details on kinetics modeling can be found in the Model tab.
References
1. Singh, S., Mulchandani, A., & Chen, W. (2008). Highly selective and rapid arsenic removal by metabolically engineered Escherichia coli cells expressing Fucus vesiculosus metallothionein. Applied and environmental microbiology, 74(9), 2924-2927.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 183
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