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.

Kinetic Modeling

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 our model can be found on our Model wiki page.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 183