Team:TecMonterrey GDL/Results
1.pOmpC/GFP
Part:BBa_R0082 was first submitted by the Antiquity 2004 team, and additional characterizations were performed by the Hamburg 2016 team. For the 2019 edition, our team will provide additional experimental data related to the influence of this part in the growth kinetics of a wild type NEB-5 alpha strain. For this, we will use a construct comprised of ompC+GFP cloned into the pSB1C3 vector to transform NEB-5 alpha cells. We will then grow our transformants in media with varying concentrations of NaCl and sucrose to evaluate their growth kinetics at high osmolarity using a standard growth curve assay.
Petri dishes observed in UV transilluminator from NEB 5-alpha E. coli cells[pOmpC-GFP].
Miniprep plasmid extraction from pOmpC-GFP in transformed NEB 5-alpha Competent E. coli cells by QIAprep® Spin Miniprep Kit. 10kb: Quick-Load Purple 2-log DNA Ladder (10kb). M(1-3): Plasmid extraction triplicate.
Culture conditions and determination of cell growth and fluorescence
E. coli was grown in Luria-Bertani broth at 37°C with vigorous shaking. When necessary, ampicillin (Amp) (Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium (100 µg/mL) Plate media were prepared by adding agar to liquid broth at a final concentration of 1.5%. Osmotic pressure conditions studied included NaCl (1, 5, 10, 20 and 40 g/L, and without NaCl), and dextrose (5 and 10%).
For the assessment of expression of GFP fluorescence during growth, the microtiter plate assay system Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA) was used. All measurements were conducted in sterile 96-well bottom microplates (Nunc, Rochester, NY, USA) with a final assay volume of 300 µl/well. Overnight cultures were diluted into fresh LB medium before growth with different appropriate experimental additives (NaCl or dextrose). The microplates were incubated for 24 h at 37°C. Measurements were made at 1 h intervals. During cultivation, the Varioskan Flash provided quantitative online data of (i) cell density via measuring OD600 and (ii) in vivo GFP expression: GFP fluorescence was monitored at 511 nm upon excitation at 488 nm.
Growth curve at OD600 at different osmotic pressure conditions.
Fluorescence curve of GFP at 488/511 nm at different osmotic pressure conditions.
Ratio based on GFP fluorescence per optical density unit at different osmotic pressure conditions.
2.pBAD/TRZ1
We designed a new part (pBAD+Trz1) to evaluate the influence of the overexpression of the Trz1 receptor (and the subsequent overstimulation of the endogenous EnvZ/OmpR system) in the growth kinetics of a wild type NEB-5 alpha strain. This construct will be cloned into the pSB1C3 vector and used to transform NEB-5 alpha cells. We will then grow our transformants in different culture media to evaluate their growth kinetics at high glucose concentrations using a standard growth curve assay.
10kb: Quick-Load Purple 2-log DNA Ladder (10kb), M1: Miniprep NEB 5-alpha E. coli cells[pBAD-TRZ1], D1: NEB 5-alpha E. coli cells[pBAD-TRZ1] digested with ECORI-HF.
3.pOmpC/TRZ1/iLOV
We designed a new part (pompC+Trz1) to evaluate the impact in cell viability of the overstimulation of the endogenous EnvZ/OmpR system, through the establishment of a positive feedback loop between the Trz1 receptor and the ompC promoter. This construct was designed as an improvement of the existing TRZ1 receptor, which is already submitted to the iGEM registry. The co-expression of TRZ1 with the fluorescent reporter iLov allows rapid screening of transformants directly on the plate as shown in the image below.
Products from Digestion & Ligation by NEB protocols. 10kb: Quick-Load Purple 2-log DNA Ladder (10kb), L1: pSB1C3 + pBAD-TRZ1, L2: pSB1C3 + pOmpC-TRZ1-iLOV.
Quick-Load Purple 2-log DNA Ladder (10kb), M1: Miniprep NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV], D1: NEB 5-alpha E. coli cells[pOmpC/TRZ1/iLOV] digested with ECORI-HF.
Representative image of fluorescent colonies corresponding to bacteria transformed with the pOmpC+TRZ1+iLov construct.
Vertical gel: In this SDS-PAGE a total protein extraction run is shown to assess the differential expression of TRZ1 in normal LB medium containing 5g/L of NaCl, and after a high osmolarity induction with LB medium containing 10g/L of NaCl.
Discussion
According to our
results, the cloning of both constructs was achieved successfully in our chassis, as
demonstrated by agarose gel electrophoresis of purified plasmid DNA for both pBAD-TRZ1 and
pOmpC-TRZ1-iLOV constructs. Representative images of the gels show bands for each construct in
the regions that correspond to their expected sizes before and after being digested with
EcoRI-HF (size of linearized plasmid: ∼4kb). Moreover, we also demonstrated the presence of the
TRZ1 protein (∼50kDa), as demonstrated by SDS-PAGE of bacteria transformed with the
pOmpC-TRZ1-iLOV construct, which were grown in normal and high osmolarity media (5 g/L and 10
g/L respectively).
To evaluate the effects of different osmolarities on bacterial growth, we performed a standard
growth curve assay with bacteria transformed with the pOmpC-GFP construct. This was done to
evaluate the effect of the stimulation of pOmpC on both bacterial growth (OD600) and reporter
expression (RFU). As shown in the OD600 growth curve, most of the growth conditions used have
similar effects on bacterial growth over time, except for 40 g/L NaCl and 10% glucose, which led
to a marked delay in the onset of the exponential growth phase. Although both conditions led to
an increase in the expression of the fluorescent reporter (GFP), the fluorescence corresponding
to bacteria growing in medium with 40 g/L NaCl decreased after 20 h. However, fluorescence
corresponding to bacteria grown in 10% glucose increased consistently until the end of the
experiment. This behavior can be seen as a large increase in the RFU/OD600 ratio at
approximately 6 h, which was mainly due to the extended lag phase. These results suggested that,
even though bacteria cultured in 10% glucose were not undergoing active cell division, their
metabolic rates were not compromised and could efficiently synthesize the fluorescent reporter.
This behavior could be explained due to the influence of the endogenous EnvZ/OmpR system, which
acted directly on pOmpC to promote the synthesis of the fluorescent reporter.
Future
Perspectives
Our future work will focus on developing a reporter system that could be more easily detected without the need for additional hardware. Our current system is designed to generate a fluorescent signal that is proportional to the concentration of extracellular glucose. Although this approach allowed us to evaluate the effect of the different transgenes and promoters that we characterized at this stage in our project, this does not enable the ease of interpretation that we envisioned. Hence, we will now focus on a different strategy based on the conditional expression of the TRZ1 receptor, and the inducible expression of an enzyme that produces a visible pigment. For this, we will couple the expression of the bpsA indigoidine synthetase (blue pigment synthetase A) from S. lavendulae ATCC11924 (Part:BBa_K1152008) to the control of pOmpC. This strategy will enable the synthesis of a visible blue pigment following the detection of increases in extracellular glucose, which would greatly facilitate the interpretation of the results by the end-user. This being said, the final objective of this project is to assemble a functional product that is capable of detecting different extracellular glucose concentrations and giving up a measurable signal controlled by inducible gene expression, namely a bio-ink that could be printed or directly applied into the skin and show the aforementioned colorimetric change due to the blue pigment expression increase according to the glucose levels of the user.
Conclusion
Highly customized strains are routinely used to generate valuable information about the roles of different proteins in the cell. However, loss of function mutations that target genes or proteins that fulfill roles that are crucial for the maintenance of cellular homeostasis could greatly impact cell viability. Furthermore, the function of several molecular targets located downstream of the affected protein could also be compromised, which often interferes with the interpretation of the results. Although, the choice of wild-type strains provides a more representative chassis for experimentation, endogenous pathways could also mask the actual function of the transgene. In this project, we aimed to develop a microorganism that could sense changes in the levels of extracellular glucose and respond by generating a detectable signal. We believe whole-cell microbial biosensors hold great promise for the development of continuous glucose monitoring systems for health care applications. For this, we chose a wild-type chassis due to the relevance of the EnvZ/OmpR system for the osmoregulatory ability of the bacteria. We characterized the ability of the ompC promoter to transduce perturbations in the biochemical environment into a readily detectable signal. We also demonstrated the inducible expression of the TRZ1 receptor upon variations in specific biochemical cues. This system holds great promise for the development of biomedical devices to monitor glucose levels in a cost-efficient, non-invasive, and versatile fashion.
