iGEM 2016 — Team Slovenia

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# Team:Slovenia/Notebook

Log Book

[![](//2016.igem.org/wiki/images/d/d1/T--Slovenia--logo.png)](//2016.igem.org/Team%3ASlovenia)

# Log book

#### Before 25.4.2016

The first team meeting was on 18.3.2016, where we discussed [initial project proposals](https://2016.igem.org/Team%3ASlovenia/Notebook/Proposals) from students accepted to the team.

Our team spent time going through literature and got together many times to brainstorm and discuss ideas. Eventually we settled on the final idea. We discussed plasmids, chemicals and cell lines already present in the Department of Synthetic Biology and Immunology, National Institute of Chemistry (Slovenia), where we spent most of these six months, performing our experiments.

![](//2016.igem.org/wiki/images/7/79/T--Slovenia--before25.4.JPG)

#### 25.4.2016 - 1.5.2016

Cloning :

* rapamycin constructs inducible split TEV protease (Nik, Tjaša)
* constructs with iRFP for retrieval in and on endoplasmic reticulum (ER) (Nik, Tjaša)
* mechanosensitive channels and gas vesicles (GvpA and GvpC) (Estera,Rok)

![](//2016.igem.org/wiki/images/8/84/T--Slovenia--25.4.JPG)

#### 2.5.2016 - 8.5.2016

Testing GvpA and GvpC:

* expression in HEK293T cell line (Estera)
* cytotoxicity in HEK293T cell line (Rok)

#### 9.5.2016 - 15.5.2016

Cloning of:

* new constructs of mechanosensitive channels and gas vesicles with coiled coil peptides (Estera, Rok)
* light inducible systems with split firefly luciferase (Nina)
* constructs for firefly luciferase with inducible reconstitution in ER via inteins (Nik)
* wild type TEV protease (Arne)
* TEV protease reporters (for wt TEV substrate, E and H variant) (Arne)
* split firefly luciferase constructs with coiled coil peptides (Katja)
* single chain calcium sensing constructs (CaMgaroo-2, SaVe2.12 and SaVe6.1) (Katja)

Testing:

* GvpA and Gvp expression in in HEK293T cell line with western blot and immunostaining (Estera)
* ratio optimization and localization with confocal microscopy (Estera)
* repeating cytotoxicity test in HEK293T cell line (Rok)
* testing construct in or on ER
* retrieval of iRFP:P3:GCN:P4:KDEL construct in ER with confocal microscopy and western blot (Nik, Tjaša)
* inducible release of from ER with confocal microscopy and western blot and immunostaining (Nik, Tjaša)

![](//2016.igem.org/wiki/images/9/97/T--Slovenia--9.5.JPG)

#### 16.5.2016 - 22.5.2016

![](//2016.igem.org/wiki/images/e/e4/T--Slovenia--16.5.JPG)

Cloning:

* cloning new constructs of mechanosensitive channels and gas vesicles with coiled coil peptides (Estera, Rok)
* light inducible systems with split luciferase (Nina)
* variant of TEV protease active in ER (Nik)
* FRET based TEV protease sensors (Arne)
* single chain calcium sensing constructs (CaMgaroo-2, SaVe2.12 and SaVe6.1) (Katja)
* permuted firefly luciferase reporter for TEV and PPV protease (Tjaša)

Testing:

* split firefly luciferase reconstitution via coiled coil interaction with dual luciferase assay (Katja)

Repeating:

* testing GvpA and GvpC expression in in HEK293T cell line with western blot and immunostaining (Estera)

#### 23.5.2016 - 29.5.2016

Cloning:

* cloning new constructs of mechanosensitive channels and gas vesicles with coiled coil peptides (Estera, Rok)
* light inducible systems with split luciferase (Nina)
* single chain calcium sensing constructs (CaMgaroo-2, SaVe2.12 and SaVe6.1) (Katja)

Testing:

* retention on ER and cleavage with recombinant TEV protease - western blot and immunostaining (Nik)
* localization of modified mechanosensitive channels and gas vesicles in HEK293T cell line with confocal microscopy (Estera)
* preparation of lipid microbubbles (Rok)
* of wild type TEV protease activity and orthogonality with the reporters with different TEV substrates (for wt TEV substrate, E and H variant)

Repeating:

* split firefly luciferase reconstitution via coiled coil interaction with dual luciferase assay (Katja)

![](//2016.igem.org/wiki/images/4/47/T--Slovenia--23.5.JPG)

#### 30.5.2016 - 5.6.2016

Cloning:

* cloning new constructs of mechanosensitive channels and gas vesicles with coiled coil peptides (Estera, Rok)
* light inducible systems with split luciferase (Nina)
* single chain calcium sensing constructs (CaMgaroo-2, SaVe2.12 and SaVe6.1) (Katja)
* iRFP transmembrane constructs with only TEVp cleavage site and ER retrieval signal (Nik)
* split firefly luciferase reporter for TEV protease (Tjaša)
* constructs with variants of TEV protease; E (TEVpE) and H (TEVpH) (Arne)

Testing:

* with ultrasound stimulation and Ca2+ imaging of mechanosensitive channels in HEK293T cell line (Estera, Rok)
* light inducible systems with luciferase with dual luciferase assay (Nina)
* TEV protease activity (Arne)
* localization of modified mechanosensitive channels in HEK293T cell line with confocal microscopy (Estera)

Repeating:

* testing Gvp expression in HEK293T cell line with western blot and immunostaining (Estera)
* preparation of lipid microbubbles (Rok)
* testing GvpA and GvpC cytotoxicity in HEK293T cell line (Rok)
* testing TEV protease activity and orthogonality with different TEV protease reporters (wt TEVp substrate, E and H variant) (Arne)

![](//2016.igem.org/wiki/images/e/e3/T--Slovenia--30.5.JPG)

#### 6.6.2016 - 12.6.2016

![](//2016.igem.org/wiki/images/b/b8/T--Slovenia--6.6.JPG)

Cloning:

* cloning new constructs of mechanosensitive channels and gas vesicles with coiled coil peptides (Rok)
* single molecule calcium sensing constructs (CaMgaroo-2, SaVe2.12 and SaVe6.1) (Katja)
* new proteases (PPVp, TVMVp) (Nik)

Testing:

* orthogonality of different variants of TEV proteases with western blot and immunostaining (Arne)
* reporters for different TEV proteases (Arne)
* reconstitution of split firefly luciferase via inteins with dual luciferase assay (Nik)
* repulsion of ER transmembrane protein construct with confocal microscopy (Nik)
* localization of constructs for retention with confocal microscopy and western blot and immunostaining(Nik)
* calcium sensing with CaMgaroo-2 (Katja)

Repeating:

* localization of modified mechanosensitive channels in HEK293T cell line with confocal microscopy (Estera)
* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels in HEK293T cell line (Rok)
* split firefly luciferase reconstitution via coiled coil interaction with dual luciferase assay (Katja)
* preparation of lipid microbubbles (Rok)
* testing of light inducible systems with split luciferase with dual luciferase assay (Nina)

#### 13.6.2016 - 19.6.2016

Cloning:

* single molecule calcium sensing contructs (SaVe2.12 and SaVe6.1) (Katja)
* light inducible systems with split protease(TEV(E) variant) (Nina)
* split protease with coiled coils peptides (Nina)
* new split proteases (PPVp, TVMVp) on rapamycin inducible system (Nik)

Testing:

* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with microbubbles and gas vesicles in HEK293T cell line (Estera, Rok)
* activity of proteases (TEVp, PPVp, TVMVp) with cpLuc reporter with dual luciferase assay (Nik)
* citotoxicity for the different TEV proteases (Arne)

Repeating:

* localization of modified mechanosensitive channels in HEK293T cell line with confocal microscopy (Estera)
* preparation of lipid microbubbles (Rok)
* localization of constructs for retention with confocal microscopy and western blot and immunostaining (Nik)
* orthogonality of different variants of TEV proteases with western blot and immunostaining (Arne)

![](//2016.igem.org/wiki/images/4/40/T--Slovenia--13.6.JPG)

#### 20.6.2016 - 26.6.2016

Cloning:

* single molecule calcium sensing constructs and two molecule calcium sensing constructs with split luciferase (Katja)
* split TEV proteases variants with adjacent peptide coils (Arne)
* split firefly luciferase with new coiled coils (Arne)
* light inducible systems with split protease(TEV(E) variant) (Nina)
* new constructs of split protease on coiled coils (Nina)
* new constructs of split firefly luciferase and antiparallel coiled coils (Nina)

Testing:

* calcium sensing with CaMgaroo-2 on live cells (Katja)

Repeating:

* calcium sensing with CaMgaroo-2 on cell lysates (Katja)
* citotoxicity for the different TEV proteases (Arne)
* localization of modified mechanosensitive channels in HEK293T cell line with confocal microscopy (Estera)
* Testing of orthogonality with different TEV proteases and TEV protease reporters with WB (Arne)
* expression of GvpA with western blot and immunostaining, with a new approach to sample treatment (Estera)
* testing of constructs for retention with western blot and immunostaining (Nik)

![](//2016.igem.org/wiki/images/3/35/T--Slovenia--20.6.JPG)

#### 27.6.2016 - 3.7.2016

Cloning:

* light inducible systems with split protease(TEV(E) variant) (Nina)
* new constructs of split protease on coiled coils (Nina)
* new constructs of split firefly luciferase and antiparallel coiled coils (Nina)
* split firefly luciferase with coiled coils (Nik)
* single chain calcium sensing contructs with split luciferase (Katja)
* two chain calcium sensing constructs with split luciferase (Katja, Nik)
* split Luc constructs with an adjacent autoinhibitory coiled coil system and TEV cleavage site between the coils (Arne)

Testing:

* localization of adjusted amounts of modified mechanosensitive channels coexpressed with gas vesicles in HEK293T cell line with confocal microscopy (Estera)
* effect of ultrasound stimulation in ultrasonic bath on transcriptional regulation (Kosta, Miha)

Repeating:

* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Estera,Rok, Kosta)
* immunostaining of membrane after WB of cell lysates which were transfected with DNA for TEVp variants and fLuc:TEVs variants to check for orthogonality of different TEV proteases (Arne)

![](//2016.igem.org/wiki/images/6/6e/T--Slovenia--27.6.JPG)

#### 4.7.2016 - 10.7.2016

![](//2016.igem.org/wiki/images/1/16/T--Slovenia--4.7.JPG)

Cloning:

* single chain calcium sensing contructs with split luciferase (Katja)
* two chain calcium sensing constructs with split luciferase (Katja, Nik)
* light inducible systems with split protease(TEV(E) variant) (Nina)
* new constructs of split protease on coiled coils (Nina)
* new constructs of split firefly luciferase and antiparallel coiled coils (Nina)
* split TEV proteases with coiled coils (Arne)

Testing:

* different concentrations of reporter for light inducible systems with split firefly luciferase with dual luciferase assay (Nina)
* intein interaction with or without scGFP repulsion signal with western blot and immunostaining (Samo, Tjaša)
* of cytotoxicity of LYN kinase (Arne)

Repeating:

* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Estera,Rok, Kosta, Miha)
* testing effect of ultrasound stimulation in ultrasonic bath on transcriptional regulation (Kosta, Miha)
* testing citotoxicity of TEV proteases’ variants (Arne)
* WB and immunostaining of cell lysates which were transfected with DNA for TEVp variants and fLuc:TEVs variants to check the orthogonality of different TEV proteases (Arne)

#### 11.7.2016 - 17.7.2016

![](//2016.igem.org/wiki/images/e/ed/T--Slovenia--11.7.JPG)

Cloning:

* single chain calcium sensing contructs with split luciferase (Katja)
* two chain calcium sensing constructs with split luciferase (Katja, Nik)
* new modified mechanosensitive channels (Estera)
* TagRFP containing transmembrane constructs for retention in and on ER (Nik)
* constructs with additional tags on protease reporter (permuted firefly luciferase)

Testing:

* destabilised coiled coils (Nina)
* different concentrations of reporter for light inducible systems with split firefly luciferase with dual luciferase assay (Nina)
* rapamycin inducible split proteases (TEVp, PPVp, TVMVp) with cpLuc reporter (Nik)
* retention of our constructs with type 2 transmembrane domain with confocal microscopy (Nik)
* reconstitution of split luciferase in lumen of ER after intein interaction with western blot and immunostaining (Samo, Tjaša)
* different protocols of sample preparation for western blotting of transmembrane proteins (Samo, Tjaša)
* reconstitution of split TEV protease via coiled coil formation with dual luciferase assay (Arne)

Repeating:

* calcium sensing with CaMgaroo-2 on cell lysates (Katja)
* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Estera,Rok, Kosta, Miha)
* testing effect of ultrasound stimulation in ultrasonic bath on transcriptional regulation (Kosta, Miha)

#### 18.7.2016 - 24.7.2016

Cloning:

* single chain calcium sensing constructs with split luciferase (Katja)
* two chain calcium sensing constructs with split luciferase (Katja, Nik)
* FRET based constructs for calcium sensing and the new modified mechanosensitive channel (Estera)
* Kozak’s sequence on protease permutated luciferase reporter (Tjaša)

Testing:

* determining localization of modified mechanosensitive channels with lower plasmid amounts in HEK293T cell line by confocal microscopy (Estera)
* coupling ability and activity of different split TEV proteases after coiled-coil dimerization. For the experiment fLuc:TEVs variants were used for reporters (Arne)
* of activity of split TEVp with immunostaining of membrane after WB. The membrane was stained with antibodies against the fLuc:TEVs reporter (Arne)
* determining localization of mechanosensitive channels and gas vesicles in 6-well plates in HEK293 cell line by confocal microscopy (Estera, Rok)
* destabilised coiled coils and light inducible system with split protease (TEV(E) variant) with dual luciferase assay (Nina)
* various amounts of proteases on corresponding cyclically permuted luciferase reporter (cpLuc) (Nik)
* transmembrane domains and retrieval signal’s functionality with fluorescent confocal microscopy after cell permeabilization and staining with antibodies (Nik, Samo)
* ER retrieval signal for the luminal proteins and its proteolytic cleavage with western blot (Samo, Tjaša)

Repeating:

* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Rok,Kosta, Miha)
* testing effect of ultrasound stimulation in ultrasonic bath on transcriptional regulation (Kosta, Miha)

![](//2016.igem.org/wiki/images/2/2c/T--Slovenia--18.7.JPG)

#### 25.7.2016 - 31.7.2016

![](//2016.igem.org/wiki/images/3/36/T--Slovenia--25.7.JPG)

Cloning:

* single chain calcium sensing contructs with split luciferase (Katja)
* two chain calcium sensing constructs with split luciferase (Katja, Nik)
* constructs for logic gates (Estera, Rok, Kosta)
* protease cpLuc reporters forTEV(E) and TEV(H) proteases (Tjaša)
* pLuc reporters for PPVp and TVMVp (Nik, Samo, Tjaša)
* new channel construct (Estera, Kosta, Rok)

Testing:

* whole and split proteases with fLuc:TEVs reporter (Nik, Samo)

Repeating:

* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Rok,Kosta, Miha)
* preparation of lipid microbubbles (Rok)
* destabilised coiled coils and light inducible system with split protease (TEV(E) variant) with dual luciferase assay (Nina)
* antibody staining of membrane after WB from previous week. The membrane was stained for fLuc:TEVs reporter after the addition split TEVp (Arne)
* repeating the experiment for different split TEV proteases, this time using fLuc:TEVs reporter variants, as well as cpLuc reporters (Arne)

#### 1.8.2016 - 7.8.2016

Cloning:

* FAS-modified channel (Estera)
* constructs for logic gates (Estera)

Testing:

* calcium sensing with single chain and two chain split luciferase constructs with dual luciferase assay (Katja)
* testing logic operations with dual luciferase assay (Estera)
* production and isolation of AP4:NeonGreen protein construct in bacteria (Kosta)
* activity of whole proteases TEVp, PPVp, TVMVp, TEV(E)p, TEV(H)p with pLuc reporter (Nik, Samo, Tjaša)

Repeating:

* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Rok,Kosta, Miha)
* different amounts of light inducible system with split protease (TEV(E) variant) with dual luciferase assay (Nina)

#### 8.8.2016 - 14.8.2016

![](//2016.igem.org/wiki/images/5/5f/T--Slovenia--8.8.JPG)

Cloning:

* constructs for logic gates (Estera)
* FAS-modified channel (Estera)

Testing:

* light inducible systems with split protease (TEV(E)) variant via western blot and immunostaining (Nina)
* activity and orthogonality of the whole proteases TEVp, PPVp, TVMVp, TEV(E)p and TEV(H)p with cpLuc and pLuc luciferase reporter (Tjaša, Nik, Samo)
* coupling of split luciferase on the autoinhibitory coiled coil system with TEV cleavage site after the addition of TEVp (Arne)

Repeating:

* calcium sensing with single molecule and two molecule split luciferase constructs with dual luciferase assay (Katja)
* production and isolation of AP4:NeonGreen protein construct in bacteria (Kosta)
* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Rok,Kosta, Miha)
* different amounts of light inducible system with split protease (TEV(E) variant) with dual luciferase assay (Nina)
* activity of splitTEV, splitPPV, splitTVMV using cpLuc and pLuc luciferase reporter (Tjaša, Nik, Samo)

#### 15.8.2016 - 21.8.2016

Cloning:

* two molecule calcium sensing system with split TEV protease (Katja)
* constructs for logic gates (Estera)
* light inducible split erTEV protease on CRY2/CIBN system (Tjaša)

Testing:

* binding between MscS:P3 and AP4:NeonGreen on the cell membrane with the confocal microscope (Rok, Kosta)
* orthogonality of the light inducible systems with split protease (TEV(E)) variant via western blot and immunostaining (Nina)
* optimization of duration of live cell incubation with rapamycin before measurement with dual luciferase assay (Nik, Samo, Tjaša)
* optimization of duration of cell lysate incubation with rapamycin before measurement with dual luciferase assay (Nik, Samo, Tjaša)

Repeating:

* calcium sensing with single molecule and two molecule split luciferase systems with dual luciferase assay (Katja)
* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with gas vesicles in HEK293 cell line - stimulation optimization (Rok,Kosta, Miha)
* light inducible systems with split protease (TEV(E) variant) via western blot and immunostaining (Nina)
* testing coupling of split luciferase on the autoinhibitory coiled coil system with TEV cleavage site after the addition of TEVp (Arne)

![](//2016.igem.org/wiki/images/1/1f/T--Slovenia--15.8.JPG)

#### 22.8.2016 - 28.8.2016

![](//2016.igem.org/wiki/images/6/6f/T--Slovenia--22.8.JPG)

Cloning:

* two molecule calcium seinsing system with split TEV protease (Katja)
* constructs for logic gates with TEVs and PPVs (Estera)
* light inducible split erTEV protease on CRY2/CIBN system (Tjaša)
* light inducible split PPVp on CRY2/CIBN system (Nik)
* autoinhibitory coiled-coil constructs with different destabilized coils and a TEVp cleavage site between the two coils. The constructs also featured nLuc (Arne)

Testing:

* coupling of split Luc adjacent to an alternative (P7 and AP8) coiled coils (Arne)
* split luciferase on different variations of the autoinhibitory coiled coil system, with a TEV protease cleavage site in between (Arne)
* ultrasound stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by luminiscence detection (Katja, Kosta, Miha, Rok)
* ER retrieval of transmembrane proteins with KKMP signal in comparison to retrieval of the ER luminal proteins with KDEL signal via western blot and immunostaining (Nik, Samo, Tjaša)

Repeating:

* testing calcium sensing with two molecule split luciferase systems with dual luciferase assay (Estera)
* testing different amounts of reporter of light inducible systems with split protease(TEV(E) variant) (Nina)

#### 29.8.2016 - 4.9.2016

Cloning:

* two molecule calcium sensing system with split TEV protease (Katja, Arne)
* constructs for logic gates (Estera)
* splitTEV\_NEGGLE, splitTEV(E) and splitTEV(H) with corresponding reporter (Nina)
* new proteases (SbMVp and SuMMVp) (Nik)
* TagRFP containing transmembrane constructs with 2 and 3 TEV protease cleavage sites (Nik)
* transmembrane construct with inducible self cleavage of retrieval signal (Nik)
* autoinhibitory coiled-coil constructs with different destabilized coils and a TEVp cleavage site between the two coils. The constructs also featured nLuc (Arne)

Testing:

* logic gates NOT A, NOT B, NOR in mammal cells (Estera)
* cleavage of the ER retrieval signal form the transmembrane protein with one tevS site with western blot via western blot and immunostaining (Samo, Tjaša)
* activity of concentration gradient of the splitTEV\_NEGGLE, splitTEV(E), splitTEV(H) with luciferase reporter with dual luciferase assay (Nik, Samo)
* testing of two different autoinhibitory coiled coil constructs with TEV protease cleavage site and split luciferase after the addition of TEV protease. We tested whether the addition of TEVp allows the reconstitution of the split Luc through CC interactions (Arne)

Repeating:

* ultrasound stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by luminiscence detection (Katja, Kosta, Miha, Rok)
* binding between MscS:P3 and AP4:NeonGreen on the cell membrane with the confocal microscope (Rok, Kosta)
* testing effect of ultrasound stimulation in ultrasonic bath on transcriptional regulation (Kosta, Miha)
* testing different amounts of reporter of light inducible systems with split protease(TEV(E) variant) (Nina)

![](//2016.igem.org/wiki/images/0/0a/T--Slovenia--29.8.JPG)

#### 5.9.2016 - 11.9.2016

Cloning:

* two molecule calcium sensing system with split TEV protease (Katja, Arne)
* logic gates constructs (Estera)
* point mutation of calmodulin sensing constructs - quickchange (Estera)
* light inducible systems with split protease (original TEV) (Nina)
* antiparallel coiled coils with split luciferase (Nina)
* splitSbMV, splitSuMMV and corresponding reporters (Samo)
* SEAP with KDEL retrieval signal (Samo)
* light inducible split PPVp on CRY2/CIBN system (Nik)

Testing:

* optimization of logic gates NOT A, NOT B, NOR (Estera)
* preparing HEK293 cells transfected with GvpA&GvpC for transmission electron microscopy (Rok)
* stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by shaking and measuring the response with luminiscence detection (Katja, Kosta, Miha, Rok)
* ultrasound stimulation with ultrasonic bath of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by luminiscence detection (Katja, Kosta, Miha, Rok)
* expression of vectors via western blot and immunostaining (Nina)
* activity of concentration gradient of the splitSbMV, splitSuMMV, splitPPV, splitTEV by dual luciferase assay (Nik, Samo)
* TEVp and PPVp with corresponding cycLuc reporters (Nik, Samo)

Repeating:

* binding between MscS:P3 and AP4:NeonGreen on the cell membrane with the confocal microscope (Rok, Kosta)

![](//2016.igem.org/wiki/images/d/dd/T--Slovenia--5.9.JPG)

#### 12.9.2016 - 18.9.2016

![](//2016.igem.org/wiki/images/c/cf/T--Slovenia--12.9.JPG)

Cloning:

* two molecule calcium seinsing system with split TEV protease (Katja)
* logic gates constructs with TEVs and PPVs (Estera)
* light inducible systems with split protease (original TEV) and antiparallel coiled coils with split luciferase (Nina)
* SEAP with transmembrane protein domain and KKMP ER retrieval signal (Nik, Samo)
* TagRFP containing transmembrane constructs with P3 coiled coil and retrieval signal (Nik)
* NeonGreen with P4 coiled coil for masking of retrieval signal (Nik)

Testing:

* autoinhibitory coiled coil constructs with split luciferase and TEV cleavage site after the addition of TEV protease (Arne)
* light and rapamycin inducible NOR gates(Estera)
* localization of modified channel P3:FAS:TRPC1:P5 with confocal mycroscopy (Estera)
* stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by drawing with glass rod and measuring the response with luminiscence detection (Kosta, Rok)
* activity of TEVp, PPVp, SbMVp, SuMMVp and their orthogonality (Nik, Samo)
* ER retrieval signal in constructs with 3 tevS sites and the construct that contains inducible self cleavage retrieval signal (Nik, Samo)

Repeating:

* ultrasound stimulation with ultrasonic bath of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by luminiscence detection (Katja, Kosta, Miha, Rok)
* stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by shaking and measuring the response with luminiscence detection (Kosta, Miha)
* binding between MscS:P3 and AP4:NeonGreen on the cell membrane with the confocal microscope (Rok, Kosta)
* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with separate GvpA and GvpC in HEK293 cell line - stimulation optimization (Rok, Kosta, Miha)

#### 19.9.2016 - 25.9.2016

Cloning:

* two molecule calcium seising system with split TEV protease (Katja)
* logic gates constructs with TEVs and PPVs (Estera)
* light inducible systems with split TEV protease (Nina)
* antiparallel coiled coils with split luciferase (Nina)
* cycLuc reporters for SbMVp and SuMMVp (Samo)
* SEAP constructs with transmembrane and ER retrieval domain (Nik, Samo)

![](//2016.igem.org/wiki/images/d/da/T--Slovenia--19.9.JPG)

![](//2016.igem.org/wiki/images/6/64/T--Slovenia--19.9b.JPG)

Testing:

* calcium sensing with two molecule system with split TEV protease with dual luciferase activity assay (Katja)
* light and rapamycin inducible NOR gates (Estera)
* localization of modified channel P3:FAS:TRPC1:P5 and calmodulin sensing constructs in HEK293T cell line with confocal microscopy (Estera, Katja)
* modified channel P3:FAS:TRPC1:P5 and calmodulin sensing constructs expression in HEK293T cell line with western blot and immunostaining (Estera, Katja)
* stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by applying pressure with a round object and measuring the response with luminescence detection. HEK293 cells were seeded on a petri dish and fixated in ultrapure agarose gel (Kosta, Rok)
* light inducible systems with split proteases (split original TEV and PPV) with dual luciferase assay and western blot and immunostaining (Nina)
* ultrasound stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by luminiscence detection (Katja, Kosta, Miha, Rok)
* orthogonality of TEVp, PPVp, SbMVp, SuMMVp with luciferase reporter with dual luciferase assay (Nik, Samo)
* splitTEV, splitTEV\_NEGGLE, splitPPV, splitSbMV and splitSuMMV with luciferase reporter with dual luciferase assay (Nik, Samo)
* ER retrieval of KDEL and KKMP with SEAP reporter (Nik, Samo)
* cleavage of the ER retrieval signal from transmembrane protein with confocal microscopy (Nik, Samo, Tjaša)

Repeating:

* experiment all the different autoinhibitory coiled coil constructs with nLuc. We tested whether the addition of TEVp allows for the reconstitution of the split Luc through CC interactions (Arne)
* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels with separate GvpA and GvpC in HEK293 cell line - stimulation optimization (Kosta, Miha, Rok)

#### 26.9.2016 - 2.10.2016

Cloning:

* contructs necessary for different logic functions (Arne)
* final constructs into BioBrick vector (Estera, Katja)

Testing:

* calcium sensing with two molecule system with split TEV protease with dual luciferase activity assay (Katja)
* rapamycin optimization for inducible NOR gates (Estera)
* ultrasound stimulation and Ca2+ imaging of mechanosensitive channels in HEK293 cell line (Kosta, Miha, Rok)
* inhibitors of calcium mechanosensitive channels in HEK293 cell line - stimulation optimization (Kosta, Miha, Rok)
* cleavage of retrieval signal from the transmembrane protein with western blot (Nik, Samo, Tjaša)
* activity and orthogonality of whole and split proteases (TEVp, PPVp, SbMVp and SuMMVp) with reporter cycLuc (Nik, Samo)
* inducible release of SEAP from transmembrane constructs via cleavage with protease (Nik, Samo)

Repeating:

* optimization of the amounts of P3:cLuc, TEVp and autoinhibitory coiled-coil constructs to get the highest possible fold increase (Arne)
* stimulation of cells containing mechanosensitive channels, gas vesicles and two molecule calcium sensing system with split luciferase by drawing with glass rod and measuring the response with luminescence detection (Kosta, Rok)
* light inducible systems with split proteases (split original TEV and PPV) with dual luciferase assay and western blot and immunostaining (Nina)

![](//2016.igem.org/wiki/images/b/bf/T--Slovenia--26.9.JPG)

#### 3.10.2016 - 9.10.2016

Cloning:

* final constructs into BioBrick vector (Estera, Katja, Tjaša)

Repeating:

* calcium sensing with two molecule system with split TEV protease with dual luciferase activity assay (Katja)

![](//2016.igem.org/wiki/images/8/88/T--Slovenia--3.10.JPG)

#### 10.10.2016 - 16.10.2016

![](//2016.igem.org/wiki/images/8/81/T--Slovenia--wiki2.JPG)

Cloning:

* final constructs into BioBrick vector (Estera, Katja, Tjaša)

Repeating:

* calcium sensing with two molecule system with split TEV protease with dual luciferase activity assay (Katja)

#### 17.10.2016 - 23.10.2016

Working on:

* wiki
* presentation
* poster
* banner

![](//2016.igem.org/wiki/images/e/e6/T--Slovenia--wiki1.JPG)

####

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