iGEM Stockholm Poster2015.igem.org/files/poster/Stockholm.pdfused anti-V5 primary antibody and...
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- Successful construction and expression of BAR - Affibody binding capacity is maintained in BAR - Coupling of the EnvZ/OmpR signaling cascade to quorum sensing 2 1 Figure 1: (A) BAR1, BAR2 and BAR3 are expressed in TOP10 E. coli upon IPTG induction. The positive and negative controls are IPTG inducible EnvZ. (B) Interaction of HER2 with BAR2 using ELISA assay. Two concentrations of HER2 were used: 3 ng/μL and 5 ng/µL. The negative control was IPTG inducible EnvZ. ns : P > 0.05 (not significant), ‘*’ : P ≤ 0.05. Figure 2: (A) Scheme of the OmpR-Regulated-RhlI construct. (B) Violacein induction in C. violaceum by E. coli transformed with OmpR-Regulated-RhlI, cultured at low and high osmolarity. ns : P > 0.05 (not significant), ‘***’: P ≤ 0.001. Early cancer diagnosis still represents one of the most crucial issues in modern medicine. We wanted to tackle this issue by creating a non-invasive, sensitive, and transferable method for early detection. For our Affibody-Based Bacterial Biomarker Assay (ABBBA), we created a new class of chimeric receptors 3 based on the bacterial osmoregulator EnvZ. We named them Bacterial Antigen Receptors (BAR). For proof-of-principle we chose to work with HER2, a breast cancer biomarker. We sometimes struggled to find relevant information on old wikis and suspected that the competitive element of iGEM was at times overshadowing the science.To estimate the extent of this issue we conducted a survey and evaluated wikis to systematically assess their structure and transparency. Expression of BAR and HER2 Binding Capacity We were able to successfully express three different Bacterial Antigen Receptors; BAR1, BAR2 and BAR3. We performed Western Blots with anti-affibody antibodies to show the expression of our BAR (Fig. 1A). We also showed interaction between BAR2 and HER2. We performed an ELISA by applying V5-tagged BAR2 on a plate coated with HER2. We used anti-V5 primary antibody and HRP-conjugated secondary antibody. There is an increased retainment for BAR2 in comparison to the two controls (Fig. 1B). OmpR Regulated Quorum Sensing We did an osmolarity bioassay with the reporter strain Chromobacterium violaceum, which produces the violet pigment ‘Violacein’ in response to quorum sensing molecules. The results show that OmpR-Regulated-RhlI (Fig. 2A) produces quorum sensing molecules in an OmpR dependent way (Fig. 2B). We used the results to write a set of guidelines: the Stockholm iGEM wiki pledge 5 . We hope that our project will change the way iGEM teams think about the importance of transparency and negative results. Upon antigen binding, the receptor would trigger the OmpR signaling cas- cade 4 , leading to the production of quorum sensing molecules. When OmpR is phosphorylated, the quorum synthase RhlI is expressed. Simulta- neously, the luxI gene is silenced by micF, a non-coding RNA. The quorum sensing molecules produced by RhlI or LuxI will respectively activate a red or green fluorescent signal in a separate readout strain. ABBBA PROJECT RESULTS PRACTICES ATTRIBUTIONS We successfully modeled endogenous EnvZ/OmpR osmoregulation in E.coli 4 . We designed and optimized a two-compartmental model with a switch that depends on the extracellular osmolarity, using MatLab and SimBiology. The model shows that changes in osmolarity regulate the endogenous OmpR dependent pathway, which controls expression of outer membrane porins. MODELING Disease Detecting Bacteria ACHIEVEMENTS Felix Richter 1 , Carmen Gallo Alvarez 1 , Denise Strand 1 , Hugo Tello 1 , Karol Kugiejko 1 , Katrine Horne Iversen 1 , Manon Ricard 1 , Naz Karadag 1 , Radoslaw Gora 1 , Sarah Wideman 1 , Shea Baxley 1 , Utsa Karmakar 1 , Hugi Asgeirsson 2 , Linnea Österberg 2 , Maximilian Karlander 2 , Mona Hassan 2 , Pontus Höjer 2 , Axel Bergenstråle 3 1 Karolinska Institute, Solna 2 Royal Institute of Technology (KTH), Stockholm 3 Stockholm University, Stockholm References 1. WHO Media Center. (2015). Key Facts About Cancer. Retrieved 23 August, 2015, from http://www.who.int/mediacentre/factsheets/fs297/en/ 2. Högbom, M., Eklund, M., Nygren, P. Å., & Nordlund, P. (2003). Structural basis for recognition by an in vitro evolved affibody. Proceedings of the National Academy of Sciences, 100(6), 3191-3196. 3. Sadelain, M., Brentjens, R., & Rivière, I. (2013). The basic principles of chimeric antigen receptor design. Cancer discovery, 3(4), 388-398 4. Cai, S. J., & Inouye, M. (2002). EnvZ-OmpR interaction and osmoregulation in Escherichia coli. Journal of Biological Chemistry, 277(27), 24155-24161. 5. iGEM Stockholm. (2015). Wiki pledge and recommendations. Retrieved 23 September, 2015, from http://2015.igem.org/Team:Stockholm/Wikipledge Supervisors and advisors Johan Rockberg, Royal Institute of Technology (KTH) Ute Römling, Karolinska Institute Teresa Frisan, Karolinska Institute Håkan Jönsson, Royal Institute of Technology (KTH) Roger Draheim, University of Portsmouth (Advisor)
Transcript of iGEM Stockholm Poster2015.igem.org/files/poster/Stockholm.pdfused anti-V5 primary antibody and...
- Successful construction and expression of BAR- Affibody binding capacity is maintained in BAR- Coupling of the EnvZ/OmpR signaling cascade to quorum sensing
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Figure 1: (A) BAR1, BAR2 and BAR3 are expressed in TOP10 E. coli upon IPTG induction. The positive and negative controls are IPTG inducible EnvZ. (B) Interaction of HER2 with BAR2 using ELISA assay. Two concentrations of HER2 were used: 3 ng/µL and 5 ng/µL. The negative control was IPTG inducible EnvZ. ns : P > 0.05 (not significant), ‘*’ : P ≤ 0.05.
Figure 2: (A) Scheme of the OmpR-Regulated-RhlI construct. (B) Violacein induction in C. violaceum by E. coli transformed with OmpR-Regulated-RhlI, cultured at low and high osmolarity. ns : P > 0.05 (not significant), ‘***’: P ≤ 0.001.
Early cancer diagnosis still represents one of the most crucial issues in modern medicine. We wanted to tackle this issue by creating a non-invasive, sensitive, and transferable method for early detection.
For our Affibody-Based Bacterial Biomarker Assay (ABBBA), we created a new class of chimeric receptors3 based on the bacterial osmoregulator EnvZ. We named them Bacterial Antigen Receptors (BAR). For proof-of-principle we chose to work with HER2, a breast cancer biomarker.
We sometimes struggled to find relevant information on old wikis and suspected that the competitive element of iGEM was at times overshadowing the science.To estimate the extent of this issue we conducted a survey and evaluated wikis to systematically assess their structure and transparency.
Expression of BAR and HER2 Binding Capacity We were able to successfully express three different Bacterial Antigen Receptors; BAR1, BAR2 and BAR3. We performed Western Blots with anti-affibody antibodies to show the expression of our BAR (Fig. 1A).We also showed interaction between BAR2 and HER2. We performed an ELISA by applying V5-tagged BAR2 on a plate coated with HER2. We used anti-V5 primary antibody and HRP-conjugated secondary antibody. There is an increased retainment for BAR2 in comparison to the two controls (Fig. 1B).
OmpR Regulated Quorum SensingWe did an osmolarity bioassay with the reporter strain Chromobacterium violaceum, which produces the violet pigment ‘Violacein’ in response to quorum sensing molecules.The results show that OmpR-Regulated-RhlI (Fig. 2A) produces quorum sensing molecules in an OmpR dependent way (Fig. 2B).
We used the results to write a set of guidelines: the Stockholm iGEM wiki pledge5. We hope that our project will change the way iGEM teams think about the importance of transparency and negative results.
Upon antigen binding, the receptor would trigger the OmpR signaling cas-cade4 , leading to the production of quorum sensing molecules. When OmpR is phosphorylated, the quorum synthase RhlI is expressed. Simulta-neously, the luxI gene is silenced by micF, a non-coding RNA. The quorum sensing molecules produced by RhlI or LuxI will respectively activate a red or green fluorescent signal in a separate readout strain.
ABBBAPROJECT RESULTS
PRACTICES
ATTRIBUTIONS
We successfully modeled endogenous EnvZ/OmpR osmoregulation in E.coli4. We designed and optimized a two-compartmental model with a switch that depends on the extracellular osmolarity, using MatLab and SimBiology. The model shows that changes in osmolarity regulate the endogenous OmpR dependent pathway, which controls expression of outer membrane porins.
MODELING
Disease Detecting Bacteria
ACHIEVEMENTS
Felix Richter1, Carmen Gallo Alvarez1, Denise Strand1, Hugo Tello1, Karol Kugiejko1, Katrine Horne Iversen1, Manon Ricard1, Naz Karadag1, Radoslaw Gora1, Sarah Wideman1, Shea Baxley1, Utsa Karmakar1, Hugi Asgeirsson2, Linnea Österberg2, Maximilian Karlander2, Mona Hassan2, Pontus Höjer2, Axel Bergenstråle3
1Karolinska Institute, Solna 2Royal Institute of Technology (KTH), Stockholm 3Stockholm University, Stockholm
References1. WHO Media Center. (2015). Key Facts About Cancer. Retrieved 23 August, 2015, from http://www.who.int/mediacentre/factsheets/fs297/en/2. Högbom, M., Eklund, M., Nygren, P. Å., & Nordlund, P. (2003). Structural basis for recognition by an in vitro evolved affibody. Proceedings of the National Academy of Sciences, 100(6), 3191-3196.3. Sadelain, M., Brentjens, R., & Rivière, I. (2013). The basic principles of chimeric antigen receptor design. Cancer discovery, 3(4), 388-3984. Cai, S. J., & Inouye, M. (2002). EnvZ-OmpR interaction and osmoregulation in Escherichia coli. Journal of Biological Chemistry, 277(27), 24155-24161.5. iGEM Stockholm. (2015). Wiki pledge and recommendations. Retrieved 23 September, 2015, from http://2015.igem.org/Team:Stockholm/Wikipledge
Supervisors and advisorsJohan Rockberg, Royal Institute of Technology (KTH)Ute Römling, Karolinska InstituteTeresa Frisan, Karolinska InstituteHåkan Jönsson, Royal Institute of Technology (KTH)Roger Draheim, University of Portsmouth (Advisor)
