Team:Stockholm/Results
Biomarker recognition
HYPOTHESIS 1: A construct of the EnvZ-Affibody chimera can be successfully expressed in E.coli
Introduction: After having modelled and synthesized our Bacterial Antigen Receptor constructs (BAR1, BAR2 and BAR3), we used western blots to show the expression of our EnvZ-Affibody chimera protein in E.coli strain TOP10. In order to check the expression of our constructs, we used antibodies targeting different parts of our chimeric receptor. The first was an anti-V5 antibody, against the C-terminal V5 tag of the EnvZ receptor. The second was an anti-affibody antibody to check the presence and expression of our BAR constructs.
Method: BAR1, BAR2, BAR3, EnvZ (WT) and the empty plasmid control were transformed in TOP10 cells. Those constructs were verified by SANGER sequencing (SUPREMERUN by GATC Biotech). Singles colonies were picked and cultures in log phase were induced overnight with 25µM IPTG. Western blots were performed using a mouse monoclonal antibody against the V5 tag (R960-CUS from Thermo Fisher Scientific) and a polyclonal goat antibody against the Affibody molecule (Goat Anti-Affibody® IgG from Affibody AB) according to the protocol [WB PROTOCOL]. For each experiment, the membrane was afterwards stripped according to the protocol and using mild conditions [STRIPPING PROTOCOL]. The membrane was then incubated with a control protein: a mouse monoclonal antibody against DnaK (ADI-SPA-880-D from Enzo). All the secondary antibodies used in these experiments were coupled to horseradish peroxidase. The imaging was acquired with a BioRad Gel DOc XR+ Imager.
Figure 1: EnvZ-Affibody chimera can be expressed in TOP10 E.coli upon IPTG induction.
Results: The three constructs and the wild type EnvZ in the IPTG inducible low copy number plasmid pRD400 have successfully been expressed (Figure 1A). The wild type as well as the constructs can be observed as homodimer with an approximate size of 100kDa (black triangle) and monomer with an approximate size of 50kDa (black circle). Further analysis, using an anti-Affibody antibody, we could confirm that only our constructs strains are expressing the desired BAR constructs (black star) (Figure 1B). The faint bands at around 25kDa (black square) are presumably caused by some unspecific binding. The loading control DnaK shows in all Western Blot lanes proofing that all wells have been loaded equally with whole cell lysates of the different strains.
Conclusion for hypothesis 1: The EnvZ-chimera proteins BAR1, BAR2 and BAR3 have successfully been expressed in E.coli TOP10.
HYPOTHESIS 2: The construct is expressed in the inner membrane
Introduction: As our chimeric receptor derives from the osmoregulator EnvZ which is situated in the inner membrane, we are suspecting that our BARs are going to have the same subcellular location. In order to confirm its localisation in the inner membrane of our chimeric receptor, we performed a FACS analysis comparing spheroplast (gram-negative cells with removed outer membrane and cell wall) to untreated cells.
Method:
Results:
Conclusion for hypothesis 2:
HYPOTHESIS 3: A construct of the EnvZ-Affibody chimera protein binds the HER2 protein
Introduction: To test whether interaction between our BAR construct and HER2 was taking place, affinity based experiments were performed. The affinity was primarily tested using column experiments to separate the BAR construct from a cell lysate. For this experiment BAR constructs 1 and 2 were tested by running them in a IMAC column with inbound recombinant HER2 which would hopefully capture the constructs through the affibody. Also, an ELISA experiment was attempted for construct 2 with HER2 fixed to the ELISA plate. Additionally a FACS analysis was performed on spheroplasts by fluorescently tagging the constructs in the inner membrane incubated with HER2.
| # | Experiments for hypothesis 3 | Status | |
|---|---|---|---|
| 1 | Can affinity be proved using affinity separation in an IMAC column? | Unknown | Go to experiment |
| 2 | Could interaction be proven using ELISA? | Positive | Go to experiment |
| 3 | Could interaction be proven for the constructs in spheroblasts using FACS? | Unknown | Go to experiment |
Experiment 1: Can affinity be proved using affinity separation in an IMAC column?
Method:
Results:
Experiment 2: Could interaction be proven using ELISA?<
Method:
Results:
Experiment 3: Could interaction be proven for the constructs in spheroplasts using FACS?
Method:
Results:
Conclusion for hypothesis 3:
HYPOTHESIS 4: A construct of the EnvZ-Affibody chimera protein is unresponsive to changes in osmolarity
Introduction: EnvZ is originally a receptor in E.coli which is sensible to osmolarity. In our three EnvZ-Affibody chimeras (BAR1, BAR2 and BAR3) a part of the periplasmic region has been replaced by the affibody. We are therefore expecting our constructs to not be reacting to changes in osmolarity. This was tested by performing osmolarity tests on our constructs.
Method: For this experiment the constructs BAR1, BAR2 were in the pRD400 backbone and transformed into E.coli EPB30. pRD400 (WT) was used as a positive control. pEB5 which is an empty plasmid in the same pRD400 backbone was used as a negative control. When the samples were in log phase, they were induced with 25µM IPTG and cultured overnight in high and low osmolarity media. Osmolarity tests were performed with triplicates and according to the protocol [OSMOLARITY PROTOCOL].
Results:
Figure X: CFP fluorescence of the different samples in low osmolarity medium (0% sucrose) and high osmolarity medium (15% sucrose).
Even if the spheroplast samples seem to be less fluorescent, the values are statistically not significantly different. Furthermore, this graph does not enable to draw any conclusions. Both spheroplasts and non-spheroplasts do not seem to be effectively induced which could be explained by a non functional strain. We were additionally unable to assess neither the presence nor the viability of the spheroplast.
Conclusion for hypothesis 4: We did not manage to prove that spheroplasts are able to express proteins and a read-out signal and cannot conclude at this point as the data was inconclusive. Further experiments should be made such as some gram-staining in order to determine the presence or not of spheroplasts.
HYPOTHESIS 5: The read-out can be activated in spheroplast E.coli
Introduction: EnvZ is expressed in the inner membrane of gram-negative E.coli and it needs to bind a protein, HER2, that cannot reach the receptor due to the outer membrane. We therefore considered the possibility to create spheroplasts by removing the outer membrane of E.coli before adding the HER2 biomarker to the sample as an attractive option. We consequently wanted to show that spheroplasts are able to express proteins and a read-out signal. We investigated this by comparing the fluorescent signal given by spheroplast and non spheroplast.
Method: In this experiment, the BL21-pBAD-GFP strain, which contains a plasmid with GFP controlled by an arabinose promoter, was used. Half of the samples were induced overnight with 6% arabinose, whereas the other half were left non induced. From the overnight cultures, half of the induced samples and half of the non induced samples were treated according to the spheroplast protocol [SPHEROPLAST PROTOCOL], whereas the other halves was left untreated. Fluorescence analysis using plate reader was then performed on all the samples at 4°C (temperature at which spheroplasts should be the most stable). The excitation wavelength used was set at 488nm and the emission wavelength at 509nm.
Results:
FigureX: Difference in fluorescence between induced and non-induced samples in both spheroplast and non spheroplast E.coli.
Even if the spheroplast samples seem to be less fluorescent, the values are statistically not significantly different. Furthermore, this graph does not enable to draw any conclusions. Both spheroplasts and non-spheroplasts do not seem to be effectively induced which could be explained by a non functional strain. We were additionally unable to assess neither the presence nor the viability of the spheroplast.
Conclusion for hypothesis 5: We did not manage to prove that spheroplasts are able to express proteins and a read-out signal and cannot conclude at this point as the data was inconclusive. Further experiments should be made such as some gram-staining in order to determine the presence or not of spheroplasts.
Intracellular signal transduction
HYPOTHESIS 6: Expression of OmpR-Regulated-RFP leads to OmpR dependent production of red fluorescence protein (RFP)
After activation of the BAR receptor, OmpR gets phosphorylated. Our first goal was to express RFP which is OmpR regulated. There was already a BioBrick (BBa_M30011) in the iGEM distribution kit that we used to fulfil our goal. As EnvZ deficient strain (BW25113 strain) does not contain EnvZ, it will not have any intracellular interference of endogenous OmpR activation with that of OmpR-Regulated-RFP. In high osmolarity, EnvZ exhibits kinase activity, leading to enormous amount of phosphorylated OmpR inside the cell which activates transcription of RFP containing gene in OmpR-Regulated-RFP [REFERENCE].
| # | Experiments for hypothesis 6 | Status | |
|---|---|---|---|
| 1 | Can we express RFP in Top10 and BW25113 E. coli bacteria? | Positive | Go to experiment |
| 2 | Is OmpR-Regulated-RFP actually OmpR dependent? | Positive | Go to experiment |
| 3 | Can we characterize OmpR-Regulated-RFP even more? | Positive | Go to experiment |
Experiment 1: Can we express RFP in Top10 and BW25113 E.coli bacteria?
Method: Top 10 and BW25113 (EnvZ deficient strain) chemo-competent E.coli bacteria were transformed with BBa_M30011 (See Transformation Protocol) after extraction from the iGEM distribution kit.
Result: RFP was successfully expressed in both the strains. Figure A shows red colonies with BBa_M30011 in BW25113 strain.
Figure A. BW25114 E. coli transformed with OmpR-Regulated-RFP (BBa_M30011).
Experiment 2: Is OmpR-Regulated-RFP actually OmpR dependent?
Method: Osmolarity test was performed in triplicates, first in TOP10 E. coli containing BBa_M30011 followed by in the BW25113 E. coli strain. Bacteria were grown in minimal media with four different concentrations of sucrose (0%, 5%, 10% and 15%). RFP was then measured in the fluorescence plate reader (Osmolarity protocol). An osmolarity experiment setup is shown in Figure B.
Figure B: Preparation of osmolarity experiment with the positive (BBa_J04450) and negative controls (BBa_K1766005) to the right.
Result: Figure C depicts osmolarity dependence of OmpR-Regulated-RFP BioBrick. TOP10 bacteria had increasing order of red fluorescence (in terms of fold change) depending on the increasing osmolarity level whereas BW25113 showed minor change in fluorescence intensity as expected.
Figure C. Fluorescence of TOP10 and BW25113 transformed with BBa_M30011 and cultured at different osmolarities. Statistical analysis was done by Student’s t-test and standard error bars of fold changes are shown in the bar diagram. Changes in the fluorescence intensities in 5% and 15% were significant in Top 10, and that of 10% and 15% were significant in BW25113. ‘*’ signifies significant P value. ns: P > 0.05 (not significant). *P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001. ****P ≤ 0.0001.
These results together show that the BioBrick is OmpR controlled and osmolarity dependent.
Experiment 3: Can we characterize Part A even more?
Method: The same BioBrick (BBa_M30011) was cloned in the low copy number plasmid (pSB3C5) on Roger Draheim's advice. This was done to have better and more detailed characterization of this part. Some deviations were made in the protocol. Here, TOP10 E. coli bacteria were grown in special osmo media (LB + Sucrose) in four replicates (See Aternative Osmolarity Protocol) instead of minimal media.
Results: The Y axis in Figure D shows average fluorescence/OD instead of fluorescence fold changes. These are compiled data from the Experiment 2 (BBa_M30011 in pSB1C3 in Top 10 strain) and Experiment 3 (BBa_M30011 in pSB3C5 in Top 10 strain). The standard deviation between the samples was much smaller in low copy number plasmid. It can be explained by the use of new osmo media as the changes were more consistent than the minimal media where the cells were under more stress. OmpR dependent change in the low copy number plasmid is less pronounced than the conventional one (pSB1C3) in this Figure. Possible explanation could be due to the composition stoichiometry in the cell (Ref). The absolute fluorescence was lower in pSB3C5 as expected. However, fluorescence fold change (compared to 0%) remained roughly the same in pSB3C5 and pSB1C3.
Figure D. Comparison between BBa_M30011 fluorescence in high (pSB1C3) and low (pSB3C5) copy number plasmid.
Conclusion for hypothesis
OmpR-Regulated-RFP was expressed in E. coli and it is characterized comprehensively. It was proven to be osmolarity dependent. The results are more stable and reliable when cultured in LB media and in low copy number plasmid.
HYPOTHESIS 7: Expression of MicF-Regulated-GFP leads to constitutive expression of green fluorescence protein (GFP) which can be silenced with MicF RNA
MicF RNA has been shown to regulate post-transcriptional expression of the ompF gene. The micF gene encodes an antisense RNA which binds to its target region in the ompF gene. This leads to inhibition of translation Ref. Taking this fact into consideration, we tried to incorporate the micF target (micF-T) and GFP in one plasmid in order to express micF regulated GFP. [REFERENCE].
| # | Experiments for hypothesis 7 | Status | |
|---|---|---|---|
| 1 | Can we express the GFP construct 1 in E.coli ? | Unknown | Go to experiment |
| 2 | Can we express the GFP construct 2 in E.coli? | Unknown | Go to experiment |
Experiment 1: Can we express the GFP construct 1 in E.coli ?
Method: Osmolarity tests were performed on TOP10 E. coli transformed with MicF-Regulated-GFP, according to ALTERNATIVE OSMOLARITY PROTOCOL. This was done to characterize the osmolarity dependence of the construct’s GFP production i.e. the micF inhibitory effect. E. coli transformed with BBa_K608011 (1) and BBa_J23106-BBa_I13504 (2) were used as positive controls. BBa_K1766005 was used as a negative control.
Results: Sequencing showed successful cloning of MicF-Regulated-GFP. Osmolarity testing showed that no significant fluorescence was achieved (See figure E).
Figure E. Relative fluorescence of E. coli transformed with MicF-Regulated-GFP cultured at different osmolarities.
Conclusion: A conclusive result that GFP is not produced made us believe that the design of the construct was faulty. After analyzing the gene we found a reading frame within the micF target gene which ran into two stop codons before reaching the GFP reading frame. We decided to try to make a fusion protein instead with the micF target included in the GFP.
Experiment 2: Can we express the GFP construct 2 in E.coli ?
Method: Figure F shows the illustration of the second MicF-regulated-GFP. To create this construct a site directed mutagenesis was performed on the first MicF-regulated-GFP. Mutagenesis primers was designed and ordered from IDT according to ARTICLE with the scars removed and the restriction site of BsmI introduced so that screening could be performed with restriction analysis. The mutagenesis protocol was made based on the product cheat recommendation on the polymerase used. The selection was enabled by the fact that the cells with the new construct had visible fluorescence.
Plasmid illustration of repaired micF-Regulated-GFP
Results: All picked colonies after transformation with the mutagenesis product showed positive result on the restriction analysis and confirmation was made by sequencing. Fluorescence was visible under UV light.
Conclusion: The only conclusion that was made is that the new construct produces fluorescence. Next step: To test if the MicF-Regulated-GFP construct actually works as intended, with the micF dependent inhibition, an osmolarity test would be conducted. The test would be performed on TOP10 E.coli as well as on the EnvZ deficient strain BW25113. Both strains would be transformed with the MicF-Regulated-GFP fusion protein in a low copy number plasmid. It could also be combined on the same plasmid with the micF gene which is ompR regulated.
HYPOTHESIS 8: Expression of OmpR-Regulated-GFP/RFP leads to OmpR dependent regulation of RFP/GFP production
Introduction: This is the final part of the fluorescence signaling system. This part is created in order to investigate both the effects of phosphorylated and dephosphorylated OmpR. The main idea is to introduce micF RNA in a plasmid, together with OmpR-Regulated-RFP and MicF-Regulated-GFP. In high intracellular osmolarity condition, micF binds to micF-T in MicF-Regulated-GFP and silences production of GFP (Red colonies are pronounced here). In contrast, GFP is pronounced in low osmolarity when micF is not binding to micF-T. Thus, it makes the whole system sensitive to both kinase and phosphatase activity in the BAR.
Methods: Osmolarity tests should be performed according to ADAPTED OSMOLARITY PROTOCOL to characterize the construct’s GFP and RFPs osmolality dependence. We want to see if the GFP production is inhibited by the OmpR dependent micF and if the RFP production is induced by phosphorylated OmpR.
Results: MicF was also cloned together with the OmpR dependent promoter in pSB1C3. The part was confirmed by sequencing but not characterized. The rest of the construction on this part was not attempted due to lack of time. Thus, it’s characterization is left for future study.
HYPOTHESIS 9: Expression of OmpR-Regulated-RhII leads to OmpR dependent expression of quorum sensing molecule BHL
Introduction: RhlI is a quorum synthase which produces the quorum sensing molecule N-butyl-homoserine lactone (BHL). In this construct RhlI is under the control of an OmpR dependent promoter. OmpR is activated when it is phosphorylated by the EnvZ receptor. In wild type E. coli this will occur at high osmolarity conditions but not at low osmolarity. To show that the OmpR-Regulated-RhlI BioBrick (BBa_K1766005) works as expected we wanted to prove that it produces BHL in an OmpR dependent manner.
Method: To show that expression of OmpR-Regulated-RhlI is controlled by OmpR we performed an osmolarity bioassay. We used Chromobacterium violaceum in soft agar as a BHL reporter. In the presence of BHL the C. violaceum lab strain CV026 produces a violet pigment called violacein. E. coli transformed with OmpR-Regulated-RhlI was cultured overnight in high and low osmolarity media. The cultures or supernatants were then applied to separate wells on the bioassay plates. E. coli transformed with RhlI generator (BBa_K082035) was used as a positive control. Untransformed E. coli was used as a negative control. [See AHL Osmolarity Dependence Protocol]
Results for supernatant plates: On all plates, weak purple pigmentation could be seen around one well. This well contained supernatant from high osmolarity cultures of OmpR-Regulated-RhlI. No purple pigmentation could be seen around the other wells.
Figure H. Spent bacterial culture supernatant applied to plates containing C. violaceum in soft agar.
Results for bacterial culture plates: Purple pigmentation could be seen around all wells except the negative control. There was visibly less pigmentation around the positive control compared to the OmpR-Regulated-RhlI samples. The diameter of the purple pigmentation around the high and the low osmolarity samples was measured and compared. The high osmolarity samples, on average, had a 23% bigger radius than the low osmolarity samples.
Left: Figure G. Bacterial cultures t applied to plates containing C. violaceum in soft agar. Right: Figure H. Violacein induction in C. violaceum. Comparison between E. coli transformed with OmpR-Regulated-RhlI, cultured at high and low osmolarity.
HYPOTHESIS 10: Expression of MicF-Regulated-LuxI leads to constitutive expression of quorum sensing molecule OHHL which can be silenced with MicF RNA.
Introduction: LuxI is a quorum synthase which produces 3-oxo-hexanoyl homoserine lactones (OHHL). In this construct LuxI has been fused to the ompF 5’UTR. This is the target for micF regulatory RNA. In wild type E. coli MicF will inhibit translation of LuxI at high osmolarity conditions. We therefore expect MicF-Regulated-LuxI to produce more OHHL at low osmolarity conditions than at high osmolarity.
Methods: To characterize MicF-Regulated-LuxI we intended to perform osmolarity tests together with an OHHL reporter strain.
Results: Due to time limitations we were unable to construct this BioBrick and could not perform the necessary experiments to prove our hypothesis. Thus, it is left for future study.
HYPOTHESIS 11: Expression of OmpR-Regulated-RhlI/LuxI leads to OmpR dependent regulation of BHL/OHHL production
Introduction: Our idea was to build this part as our final product in the signaling pathway. The theoretical goal was to have more production of BHL in high osmolarity condition and more OHHL production in low osmolarity condition in the same bacteria, similar to OmpR-Regulated-GFP/RFP.
Results: Due to limited time of the project, we could not design or perform the necessary experiments to prove our hypothesis. Thus, it is left for future study.