Team:HKUST/Experiments

ShellBi

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Experiments

Let's Get Our Hands Wet!

Overview

Our experiments are meant to test out the genetic machinery and how different fragments may interact with one another. Due to time constraints and the fact that the error rate increases with circuit complexity, we gravitated towards characterising individual parts first. The complete proposed solution requires engineering chassis as identified through a phylogenetic tree (See dry lab) but at our stage, we wished to test out the gene interaction before the incorporation of the whole circuit in those species. Moreover, we require access to neurotoxic drugs in order to test the function of ShellBi, however, the Hong Kong regulations do not permit their use, neither is our laboratory equipped to handle such drugs. Therefore, in our experiments, we try to replicate the scenario in the closest manner possible.
Our experiment plan involves 5 major facets:

• ❒ Extracellular vs. Intracellular osmolarity
• ❒ Characterization of Two Component System (TCS) Basal using Fluorescent Assay
• ❒ Characterization of the Antisense Construct, OmpC, and OmpF promoters
• ❒ Significance Testing for the Scaffold
• ❒ Significance of F4 Negative Regulator on OmpF Promoter

How Do We Clone Our Construct?

General Molecular Cloning Strategy

All DNA fragments ordered from IDT or Twist BioScience are flanked with biobrick prefix and biobrick suffix sequence. To test each construct, every DNA fragment was amplified using Q5 High-Fidelity DNA Polymerase with prefix forward and suffix reverse overhang primers unless stated otherwise. Thereafter, the PCR product is run through a 0.8% agarose gel to verify the size of the PCR product and to exterminate the secondary products resulting from non-specific binding of the primers. After the PCR product is run through a gel, and the size is verified, the band will be purified using Thermo Scientific GeneJet Gel Extraction Kit.

To assemble the construct, we cut the purified DNA fragment using EcoRI and PstI restriction enzymes and ligate it with the pSB1C3 backbone which has been cut by the same enzymes. Transformation to BL21 strain and colonies were done subsequently. In our project, a BL21 Escherichia coli strain is used since we want a high expression of the fluorescent proteins.

Once we get the transformed colonies on a chloramphenicol plate, colony PCR will be performed on selected colonies to further verify the construct. For the colony PCR, a set of forward and reverse primers called VF2 and VR which anneals to regions flanking the prefix forward and suffix reverse. Post colony PCR, the product is run on 0.8% agarose gel to see the amplified fragment. However, since the primers don’t anneal to the insert, when running it on the gel, we should expect an extra ~100 bp sequence from the original insert length.

When the band size observed from the gel electrophoresis is claimed to be correct, the corresponding colony can be inoculated in lysogeny broth (LB) medium overnight with chloramphenicol as the selection agent. Post inoculation, the plasmid will be extracted using Thermo fisher GeneJET Plasmid Miniprep kit and the plasmid will be used for downstream experiments.

Our Experiment Plan Involves 5 Major Facets

Extracellular vs. Intracellular Osmolarity

Aim

Since we were unable to obtain neither of the toxins, we would like to replicate the effect of veratridine and shellfish toxins which induces intracellular osmolarity change.

Experimental design

To replicate the effect, we inoculated wild-type BL21 E.coli strain in lysogeny broth (LB) medium with different sodium concentrations. Since E.coli responds to osmotic challenges by regulating solute influx, the intracellular osmolarity of the cell changes with respect to the extracellular osmolarity which is the medium in which we grew the cells [1]. After inoculation, we normalized the cell number, washed the cells with MilliQ, sonicate the cells to release the intracellular osmolytes, and take osmometer measurements.

Characterization of Two Component System (TCS) Basal using Fluorescent Assay

Aim

In this construct, mRFP1 is controlled under an OmpF promoter which works in low intracellular osmolarity. While eGFP is controlled under an OmpC promoter which works in high intracellular osmolarity. With the fluorescent assay, we would like to validate our circuit and characterize the promoters by looking at the intensity of green and red fluorescent proteins. Green fluorescent color should be observed under high osmolarity and red fluorescent color should be observed under low osmolarity.

Figure 1: Cloning the TCS basal fragment

Experimental design

The TCS basal construct is generated with the general molecular cloning method aforementioned. In order to characterize the promoter and observe whether or not it is working as per literature, we inoculated the TCS basal clones in each LB medium of different sodium concentrations. To measure variation in the experiment such that statistical tests can be performed to evaluate the difference, we inoculated 2 biological replicates for each sodium concentration of LB medium.
Sodium Concentration in LB Medium

Table 1: Sodium concentrations of LB medium

After overnight inoculation, we transfer 100uL of the culture to a 96 well plate for fluorescent measurements using the plate reader. In order to visualize the green and red fluorescent proteins, we excite the sample with wavelengths of 488 nm and 584 nm respectively.

Characterization of the Antisense Construct, OmpC, and OmpF promoters

Aim

Since we wanted a distinct red or green color as the indicator of our biosensor, we designed it to be more specific by adding an antisense structure. We would like to see whether or not the antisense is significant in reducing the opposite color. Furthermore, since the antisense fragment is regulated under OmpF/C, this assay can also illustrate how the promoter responds to varying intracellular osmolarity. Since the eGFP antisense is controlled under the OmpF promoter, thus, we should see a higher eGFP repression in lower osmolarity. On the other hand, mRFP antisense is controlled under OmpC promoter, therefore, we should observe a higher activity of the antisense in higher osmolarity.

eGFP antisense, OmpF promoter

Figure 2: Cloning the constitutive eGFP + eGFP under PompF and constitutive eGFP as the control

eGFP antisense, OmpF promoter - Experimental design

For obtaining the eGFP - eGFP antisense construct, we performed the general molecular cloning method to obtain the construct. However, since we wanted an eGFP construct without the eGFP antisense, we ordered a primer which anneals between the OmpF promoter and the eGFP gene terminator to obtain the eGFP fragment.

To test whether or not the antisense is significant in reducing the opposite color, we inoculated eGFP - eGFP antisense construct in each sodium concentration of LB medium aforementioned (Table 1) with 2 biological replicates for each concentration. As a control, we also inoculate the eGFP construct with the same manner as the eGFP - eGFP antisense. The eGFP construct without the eGFP antisense was set as the reference and observation as to whether the eGFP expression is reduced by the antisense can be detected using a plate reader. To excite the eGFP, we set the wavelength to 488 nm.

mRFP1 antisense, OmpC promoter

Figure 3: Cloning the constitutive mRFP1 + mRFP1 antisense under PompC and constitutive mRFP1 as the control

mRFP1 antisense, OmpC promoter - Experimental design

As for the mRFP1 - mRFP1 antisense, a general molecular cloning method was performed to obtain the construct. A similar logic was also applied to obtain the mRFP construct without the mRFP antisense. We utilize a reverse primer between the OmpC promoter and the mRFP1 gene to obtain the mRFP construct without .

To compare the red fluorescent intensity with and without antisense, we inoculated both constructs which are the mRFP1 with mRFP1 antisense and the mRFP1 without the antisense in each sodium concentration of LB medium (Table 1) with 2 biological replicates For each concentration. The red fluorescence intensities of both constructs in each sodium concentration of LB medium will then be compared to imply the significance of the antisense and OmpC promoter.

Significance Testing for the Scaffold

Aim

At the onset, we decided to incorporate the scaffold mechanism to increase the interaction of OmpR and EnvZ. To utilize the scaffold system, it requires us to engineer the native EnvZ and OmpR such that their interaction can be enhanced with their association with the scaffold. Furthermore, engineering the EnvZ also increases the circuit’s sensitivity due to its novel auto-inhibition ability. For more details, refer to our design page.

Design page

The enhanced interaction of EnvZ and OmpR should theoretically result in an increased fluorescent protein intensities. Besides, there should be a clearer cut off between the appearance of green fluorescent and red fluorescent as a result of EnvZ auto-inhibition. Thus, to test whether or not the scaffold mechanism and engineered EnvZ increase the fluorescent intensity and accuracy, we conducted a fluorescent assay to observe the change with respect to the normal circuit without the scaffold mechanism.

Figure 4: Cloning the TCS basal with scaffold and the TCS basal only as the control

Experimental design

The scaffold is controlled under an OmpC promoter such that it is expressed under high osmolarity. That is, because we want to maintain the low rate of phosphotransfers from EnvZ to OmpR so that OmpF can have a stable expression while the OmpC expression can be inhibited.

The TCS and the scaffold (TCS scaffold from hereinafter) were ordered in the same fragment from IDT. For the scaffold to work, we would need the engineered EnvZ and OmpR on the same construct. The ligation of the 2 fragments via RCF10 method to the pSB1C3 backbone is still under progress. As the control, we also have the non engineered EnvZ and OmpR ligated to the TCS basal and pSB1C3 as one whole construct (without the scaffold).

Wavelengths of 488 nm and 584 nm to excite the green FP and red FP respectively were set. Subsequently, the plate reader result between the control and the engineered EnvZ - engineered OmpR - TCS scaffold construct will then be compared.

Significance of F4 Negative Regulator on OmpF Promoter

Aim

The OmpF promoter that we used in the context of our project, will be expressed at low intracellular osmolarity conditions. While at high intracellular osmolarity conditions, the OmpF promoter activity will be repressed. The reason being, is that the OmpF promoter that we utilize is equipped with 4 different regulatory domains, refer to below link for more information. F1, F2, F3 have high affinity binding to OmpR~P. Thus, even at low concentrations where there are low copies of OmpR~P, the OmpR~P will still bind to the F1, F2, and F3 sites to turn the gene on. In addition to the 3 activation domains, there’s also an F4 site, which has a low affinity binding site to OmpR~P. Thus, at higher concentrations where there will be higher copies of OmpR~P, the OmpR~P will occupy the F4 site and the OmpF gene expression will be repressed. With this experiment, we want to analyze the significance of the F4 negative regulator site on the OmpF promoter. Hypothetically, at higher concentrations, PompF equipped with the F4 site would have a lower expression of the mRFP1. While the PompF without the F4 site would have a higher expression of mRFP1 at higher concentrations.

Design page

Figure 5: Amplification of PompF with the F4 site and mRFP1

Figure 6: Amplification of PompF without the F4 site and mRFP1

Experimental design

To test the significance of the F4 site on PompF, we would have to generate a construct of PompF with and without the F4 attached to mRFP1 as the reporter. To obtain the 2 constructs, we used TCS basal as the template and designed the primers accordingly.

To get the PompF (+F4 site) - mRFP1 construct, we used a forward primer with a biobrick prefix overhang that anneals to the sequence upstream from the F4 site and a suffix reverse primer. While to obtain the PompF (-F4 site) - mRFP1 construct, we designed a forward primer that would anneal to the sequence upstream from the F1 site that also has a biobrick prefix overhang and a suffix reverse primer.

Ideally, after getting the 2 constructs, we would inoculate both constructs in each sodium concentration of LB medium (Table 1) with 2 biological replicates for each concentration. Afterwards, the red fluorescence intensity would be measured using a plate reader with an excitation wavelength of 584 nm. However, the cloning of PompF (with the F4 site) - mRFP1 construct is still under progress.

References:

1. Wood, Janet. “Bacterial Responses to Osmotic Challenges.” Digital Object Identifier System, Journal of General Physiology, 13 Apr. 2015.

PROJECT

• Description
• Design
• Engineering
• Proof of Concept
• Implementation
• Safety
• Contribution
• Notebook

WETLAB

• Experiments
• Results

DRYLAB

• Model

REACH

• Human Practice
• Collaboration
• Communication
• Education
• Sustainable

PARTS

• Basic Parts
• Composite Parts

TEAM

• Team
• Attributions