http://2009.igem.org/wiki/index.php?title=Special:Contributions/Ashley&feed=atom&limit=50&target=Ashley&year=&month=2009.igem.org - User contributions [en]2024-03-29T09:51:18ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Brown/Notebook_weekly_Logs/Weekly_Team2_NotebookTeam:Brown/Notebook weekly Logs/Weekly Team2 Notebook2009-10-22T03:34:39Z<p>Ashley: /* Histamine Sensor Weekly Lab Log */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor Weekly Lab Log=<br />
<br />
<br />
<br />
Week 6<br />
----<br />
<br />
July 20, 09<br />
<br />
Plan for the week:<br />
*Run gels: digest OmpC, TetA<br />
*gel purify<br />
*ligate Tet into pGEMT-easy→ DH5α transformation<br />
*ligate OmpC-TetA standard assembly→ DH5α transformation<br />
*OmpR BB: grow cultures tonight→ genomic purification tomorrow<br />
*PCR Taz1BB<br />
*Amp-Kan-Tet plates<br />
*Mutagenic PCR<br />
<br />
Tasks accomplished today:<br />
<br />
1) Ashley did PCR of Taz1<br />
*BioBrick Primers: Tar BB For (19.3 nm); EnvZ Rev (17.1 nm)<br />
*Control Primers: Amp Taz 1 For (23.0 nm); Control Taz1 Rev (26.9 nm)<br />
(i) Resuspend dry primers to 100 μM stock<br />
(ii) Make 20 μM working stock ( 1μL 100 μM stock + 4 μL H2O= 5 μL total)<br />
(iii) Template: Taz1 plasmid (miniprep)<br />
<br />
*Mastermix: 47 μL<br />
*Primer For + Rev: 1 μL each<br />
*3 tubes of BioBrick, 3 tubes of control<br />
<br />
*PCR program: <br />
1) 94°C for 5 min<br />
2) 94°C for 30 sec<br />
3) 57°C* for 30 sec<br />
4) 72°C for 1.5 min<br />
5) GoTo 2, 34 times<br />
6) 72°C for 5 min<br />
7) 4°C forever<br />
<br />
2) Gel results for digests: <br />
<br />
*1% gel<br />
<br />
**L1: 1 kb ladder<br />
**L 2: Tet Steph (EcoRI, XbaI)<br />
**L3: Tet MC ( E,X)<br />
**L4: Tet Ash (E,X)<br />
**L5: Tet Steph (E,P)<br />
**L6: Tet MC (E,P)<br />
**L7: Tet Ash (E,P)<br />
**L8: Tet old (E,P)<br />
<br />
<br />
<br />
<br />
<br />
<br />
*2% gel<br />
<br />
**L1: 1 kb<br />
**L2: Omp2 MC<br />
**L3: Omp1 Steph<br />
**L4 : Omp 2 Steph<br />
**L5: 100 bp ladder<br />
**L6: 100 bp ladder<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
*Did gel extraction on all samples<br />
<br />
3) Making competent RU1012<br />
<br />
(i) Plated RU1012 agar stab on LB-Kan plate<br />
(ii) 11 am: started liquid culture<br />
(iii) 6 pm: inoculate into SOB broth (2 mL, 4 mL, and 10 mL samples)<br />
(iv) check OD600 with 3 μL sample on nanodrop<br />
<br />
*11:40 am: <br />
**2 mL: 0.356<br />
**4 mL: 0.380<br />
**10 mL; 0.388<br />
<br />
4) Testing transformation efficiency of DH5α<br />
(i) add 50 μL competent cells + 1 μL plasmid (OmpR miniprep 7a)<br />
(ii) incubate 30 mins on ice<br />
(iii) heat shock 30 sec at 42 °C <br />
(iv) incubate on ice for 2 min<br />
(v) 200 μL LB media<br />
(vi) incubate 20 min at 37°C <br />
(vii) plate different densities: 100 μL, 10 μL, 1 μL, incubate at 37°C at 4:30 pm<br />
(viii) calculate CFU<br />
<br />
5) Gel for Taz1<br />
<br />
*Lane 1: 1 kb ladder<br />
*Lane 2: 100 bp ladder<br />
*Lane 3: Taz 1<br />
*Lane 4: Taz 2<br />
<br />
<br />
<br />
July 21, 09<br />
<br />
Tasks for today: <br />
*Run digest again, test E and S on tet<br />
*Genomic purification of DH5α then PCR OmpR, Run gel<br />
*PCR Taz1, Run gels<br />
*Calculate competency of gels<br />
<br />
1) Digest of Tet (from 7/16 minipreps) with EcoRI and SpeI<br />
<br />
*Concentration (ng/μL ), μL DNA, μL water<br />
**Tet Steph: 129.3, 7.737, 8.27<br />
**Tet Ash: 126.1, 7.93, 8.07<br />
**Tet Michael: 71.8, 13.93, 2.07<br />
<br />
Incubate till 12:00 pm (1.5 hours)<br />
<br />
<br />
*Lane1: 1 kb ladder<br />
*Lane 2: Ash<br />
*Lane 3: Ash 2<br />
*Lane 4: 1 kb ladder<br />
*Lane 5: Steph<br />
*Lane 6: Steph 2<br />
*Lane 7: Michael<br />
*Lane 8: 1kb<br />
<br />
*E, S cut successfully for Tet→ enzymes are ok<br />
<br />
2) Genomic purification performed.<br />
*Primers: OmpR BB Reverse, OmpR BB Forward<br />
*Mastermix: 47 μL; Primers: 1 each, DNA: 1 μL <br />
<br />
<br />
*Lane1: 1 kb ladder<br />
*Lane 2: 1 kb ladder<br />
*Lane 3: Tube 1<br />
*Lane 4: Tube 2<br />
*Lane 5: Tube 3<br />
*Lane 6: Tube 4<br />
*Lane 7: 100 bp ladder<br />
*Lane 8: 1kb<br />
<br />
*Do not use tubes 2 and 3 of genomic DNA<br />
<br />
3) PCR of Taz 1<br />
<br />
<br />
*A: amplification. B: Biobrick; Numbers correspond to Taz samples from minipreps of 7/17)<br />
<br />
*Gel 1 <br />
**Lane 1: 1kb<br />
**Lane 2: 1A<br />
**Lane 3: 1B<br />
**Lane 4: 2A<br />
**Lane 5: 2B<br />
**Lane 6: 3A<br />
**Lane 7: 3B<br />
**Lane 8: 1 kb<br />
<br />
*Gel 2:<br />
**Lane 1: 1kb<br />
**Lane 2: 4A<br />
**Lane 3: 4B<br />
**Lane 4: 5A<br />
**Lane 5: 5B<br />
**Lane 6: 6A<br />
**Lane 7: 6B<br />
**Lane 8: 1 kb<br />
<br />
July 22, 09<br />
<br />
1) OmpC: Grow liquid cultures, make glycerol stocks, digest with EcoRI and SpeI, run 20 μL on 3% gel with minimal loading dye<br />
<br />
2) Sequencing: OmpR, Registry (purified plasmid form), purifying PCR product<br />
<br />
3) Sequencing, Registry Taz1→ miniprep transformation in DH5α; make glycerol stocks; purify PCR<br />
<br />
*Purification of OmpR1, Taz 2B, Taz 4A<br />
*Nanodrop concentrations:<br />
**OmpR1: 28.1 ng/μL<br />
**Taz 2B: 16.6 ng/μL<br />
**Taz 4A: 16 ng/μL<br />
<br />
*Sent in for sequencing <br />
<br />
4) tetR: ligation into pGEM T easy (EcoRI, PstI)→ ligation into appropriate expression vector<br />
<br />
*Digest: 8μL pGEM, 8 μL water, 2 μL multicore buffer, 1 μL EcoRI, 1 μL PstI<br />
*Incubate at 37°C <br />
<br />
*Ligation of TetA into pGEM T-easy<br />
**10x buffer 2μL<br />
**ligase 1 μL<br />
**vector 1μL<br />
**DNA 10 μL<br />
**Water 6 μL<br />
**Incubate overnight at 4°C<br />
<br />
July 23, 09<br />
<br />
1) 9am: stop inoculation of ompC, miniprep, nanodrop, sequencing, glycerol stocks<br />
2) Find expression vector for tetR and ligate (from pGEM T-easy-tetR ligation)<br />
3) Design primers for ompC<br />
4) Make RU1012 competent <br />
<br />
Taking concentrations of RU1012 00 λ600<br />
*10:30 am 0.01<br />
*11:30 am 0.03<br />
*12:30 pm 0.05<br />
*1:10 pm 0.09<br />
*2:00 pm 0.12<br />
*2:40 pm 0.15<br />
*3:10 pm 0.2<br />
*3:50 pm 0.26<br />
*4:20 pm 0.33<br />
*4:50 pm 0.38<br />
*5:20 pm 0.50<br />
<br />
*Completed Inouye protocol, stored at -80°C<br />
<br />
<br />
July 24, 09<br />
<br />
1) Inoculated tet-pGEM T-easy in liquid cultures→ miniprep, nanodrop, digest→ expression vector<br />
2) Run gel: <br />
**Omp: cut with S, P<br />
**Tet: cut with X,P<br />
**Plasmid: 2079 bp<br />
**Tet: 1191 bp<br />
**OmpC: 108 bp<br />
<br />
*Supercoiled v.s Linear v.s Circular plasmids<br />
*Supercoiled: naturally produced by E.coli (i.e. miniprep) runs faster than linear plasmid<br />
*Linear: plasmid fom restriction digest<br />
*Circular: covalently closed plasmid (i.e ligation) runs slower than linear plasmid<br />
<br />
July 25, 09<br />
<br />
1) Standard assembly<br />
**10 μL Ligation mix<br />
<br />
2) Gel Extraction<br />
3) Nanodrop Concentrations:<br />
**OmpC1: 1.8 ng/μL<br />
**OmpC2: 3.9 ng/μL<br />
**Tet3: 12.3 ng/μL<br />
**Tet1: 4.6 ng/μL<br />
<br />
4) Digest OmpC with SpeI and PstI<br />
*Nanodrop concentration:<br />
**Omp1 MC: 30.8 ng/μL<br />
**Omp2 MC: 31.1 ng/μL<br />
<br />
5) Ligate Tet3 into OmpC digest<br />
6) Transformation of OmpC-TetA into DH5α and RU1012<br />
<br />
Syzmanski Protocol<br />
*50 μL cells and 1 μLDNA→ ice for 30 mins<br />
*Heat shock 42°C for 30 sec<br />
*2 min on ice<br />
*200 μL LB<br />
*incubate 20 min at 37°C<br />
*plate on Amp plates<br />
<br />
July 26, 09<br />
<br />
1) Inoculated ompC-tetA ligations @ 9:30 am→ miniprep liquid cultures→ glycerol stocks<br />
<br />
<br />
<br />
Week 7<br />
----<br />
<br />
July 27, 09<br />
<br />
1) Digests: RBS (SpeI, PstI), TetA (XbaI, PstI), OmpC (SpeI,PstI), TetA-pGEM (XbaI, PstI)<br />
2) Ligation with Digested Tet from July 24th<br />
3) Transform ligation: RBS-tetA→ inoculate ligation→miniprep, glycerol stocks<br />
4) Ran gel: tet-pGEM (digest with X< P); should see 2 bands at 3000 and 1000 bp→ gel purify tetA<br />
5) Digest: pBluscript (160.7 ng/μL)--? Transform into DH5α→ tet plates<br />
<br />
July 29, 09<br />
<br />
1) Digest SK with X, P<br />
2) Obtain Tet X,P→ ligate into SK<br />
**sequencing<br />
**ligation with RBS (done with optimized protocol and standard protocol, incubated at 4°C overnight<br />
<br />
3) Make amp-tet plates<br />
4) Transform RU1012 with Taz1 (incubation started at 4:40 pm)→ run SDS page gel<br />
<br />
August 2, 09<br />
<br />
1) IPTG induction of Taz1 from RU1012<br />
**8:30 pm: started liquid cultures (5 mL LB, 5 μL Amp, colony of RU1012+Taz<br />
**10:30 am: move 0.5 mL liquid culture to 5 mL new liquid culture<br />
**IPTG volumes: 5 μL, 6 μL, 7.5 μL, 10 μL<br />
**12:30 pm: spun 1 mL aliquot of 6 μL and 7.5 μL liquid cultures, spun 5 mL aliquot of 5 and 10 μL liquid cultures→ freezer<br />
**1mL cultures<br />
***resuspend in 200 μL dH2O<br />
***take 20 μL of dH2O and move to another tube<br />
***add 2x sample buffer<br />
***incubate 5 min at 95 °C, vortex, lyse<br />
***spin at high speed, load supernatant<br />
<br />
<br />
<br />
Week 8<br />
----<br />
<br />
<br />
August 4, 09 <br />
<br />
1) Tested Mutagenic Primers<br />
2) SDS Page of Taz1→ run for 90 min at 121 V, stain overnight<br />
3) DNA purification of PCR TazMut<br />
4) Ligation of purified PCR product: mut Taz into pGEM<br />
<br />
August 5, 09<br />
<br />
1) Ligation of OmpR BB and Taz1 BB into pGEM T-easy<br />
2) Transformation into DH5α, plate on Amp plates<br />
**ligation was unsuccessful→ redo<br />
<br />
August 6, 09<br />
<br />
1) Redo transformations<br />
**Control: weird, clear colonies<br />
**Taz MutC: 2 colonies<br />
**Taz MutE: some clear colonies<br />
**Taz MutD: good plate<br />
**Taz MutA: none<br />
**Taz Mut B: some<br />
**Taz BB: some<br />
**OmpR BB: good plate<br />
<br />
2) liquid cultures of Taz B,C,D,E, Taz BB and OmpRBB<br />
3) miniprepped all but TazE (nothing grew)<br />
4) Sent in mutagenic products for sequencing<br />
<br />
<br />
<br />
Week 9<br />
----<br />
<br />
<br />
August 10,09<br />
<br />
1) Digests<br />
a. pGEm-OmpR with E,P→ insert<br />
b. pGEM-Taz with E,P-→ insert<br />
c. July 11 miniprep samples of OmpC to get BB ector with E,P cut sites→ vector backbone<br />
d. Ran a gel→ gel extraction<br />
e. Revived glycerol stocks of Taz1<br />
f. Inoculated July 27 Taz1-DH5α transformation colonies in liquid cultures<br />
<br />
August 11, 09<br />
<br />
1) Round the Horn PCR!<br />
<br />
Primers:<br />
(i) P (phosphorylated)-For Mut R1 Taz 1+ Rev Mut R1Taz1-P<br />
(ii) P-For Mut R2 Tot Taz1+ Rev Mut R2 Tot Taz1-P<br />
(iii) For Mut R2 A Taz1+ Rev Mut R2 Tot Taz1-P<br />
(iv) P-For Mut R2B Taz1+ Rev Mut R2B Taz1-P<br />
(v) ForMut R1 Taz1+ Rev Mut R1 Taz1 (linear)<br />
(vi) For Mut R2B Taz1+ Rev Mut R 2B Taz1 (linear)<br />
<br />
Primer Phosphorylation:<br />
(i) 37 μL H2O<br />
(ii) 5 μL PNK buffer<br />
(iii) 1 μL 50mM MgSO4<br />
(iv) 5 μL primers (100 μM)<br />
(v) incubate at 37°C<br />
(vi) kill PNK- heat mixture at 95°C for 5 min<br />
<br />
August 12, 09<br />
<br />
1) started PCR<br />
<br />
PCR mixture<br />
*39 μL dH2O<br />
*5 μL 10x polymerase buffer<br />
*1.5 μL forward primer<br />
*1.5 μL backward primer<br />
*1 μL DNTPs<br />
*1 μL template=miniprep Taz1<br />
*1 μL Pfx platinum polymerase<br />
<br />
PCR program<br />
(i) 95°C 1 min<br />
(ii) 93 °C 30 sec<br />
(iii) 48 °C 30 sec<br />
(iv) 72 °C 18 sec<br />
(v) Go to 2 25 times<br />
(vi) 4 °C hold<br />
<br />
2) Loren Looger’s suggestions for changing Tar to become histidine receptor<br />
*R69E, R69D, R69Q, R69N<br />
*R73E, R73D, R73Q, R73N<br />
<br />
August 13, 09<br />
<br />
*Ligation of PCR products<br />
<br />
August 14, 09<br />
<br />
1) PCR amplification of Taz with new primers<br />
2) Gel visualization (bright bands!)<br />
3) Gel extraction (Qiagen)<br />
4) Ligation<br />
<br />
August 15, 09<br />
<br />
1) Transformation results: no colonies on all except Taz1 PCR (150 μL ) and Taz Gel (50 μL )→liquid cultures<br />
2) Ligation again with different optimized combinations→ good results<br />
<br />
August 16, 09<br />
<br />
1) miniprep of Taz-pGEM ligations<br />
2) Transformation of Taz-pGEm ligations<br />
<br />
<br />
<br />
Week 10<br />
----<br />
<br />
<br />
August 17, 09<br />
<br />
1) PCR Biobrick Taz→ run gel→ extract→ ligation into pGEM<br />
<br />
*20μL H2O<br />
*25 μL mastermix<br />
*1 μL template Taz miniprep 5 from 8/11<br />
*2 μL forward BB primers<br />
*2 μL reverse BB primers<br />
<br />
PCR program: <br />
(i) 94°C 5 mins<br />
(ii) 94 °C 30 sec<br />
(iii) 53.5 °C 30 sec<br />
(iv) 72 °C 90 sec<br />
(v) Repeat 2-5 34x<br />
(vi) 72 °C 5 mins<br />
(vii) Hold 4 °C <br />
<br />
*Nanodrop concentrations of gel extractions of Taz BB PCR:<br />
**Taz BB PCR1: 57.2 ng/μL<br />
**Taz BB PCR2: 66.7 ng/μL<br />
<br />
2) Digest Taz 1 minipreps (pGEM) wih Bam and Eco→ gel extract<br />
<br />
*Taz 1 miniprep (8/16 samples): Nanodrop concentrations (ng/μL) <br />
(1) :95.70 (2): 89.64 (3): 164.52 (4): 150.93 (5): 198.37 (6): 76.46 (7): 86.61 (8): 99.79<br />
Digest at 37°C for two hours. 11:40 am-1:40 pm<br />
<br />
3) Ligate Taz 1 into pGEm<br />
<br />
*Use Team 1’s gel extraction of pGem backbone(17.5 ng/μL), digested with B, E<br />
*Ligate digested Taz1 into pGem: <br />
**3 μL insert, 1 μL vector<br />
**1 μL ligation buffer (10x)<br />
**1 μL ligase<br />
<br />
*Use optimized gel extraction protocol: load 2 20 μL samples in separate wells<br />
*Cut as close as possible for each band→ combine in 1 tube<br />
*QiaQuick Protocol notes: no unnecessary steps<br />
<br />
4) Liquid culture at 5 pm of Taz1-pGem ligations<br />
5) Geneart order<br />
<br />
<br />
August 18, 09<br />
<br />
1) Miniprep of Taz-pGEM<br />
2) Run gel of digest of Bam-Eco on Taz-pGEM→gel extract→ ligation into pNoTat<br />
<br />
<br />
*Lane 1: 1 kb ladder <br />
*Lane 2: digest #1<br />
*Lane 3: #2<br />
*Lane 4: -<br />
*Lane 5: #3<br />
*Lane 6: #4 (mixture wasn’t 20 μL)<br />
*Lane 7: -<br />
*Lane 8: #5<br />
<br />
*Lane 1: 1 kb ladder <br />
*Lane 2: -<br />
*Lane 3: #6<br />
*Lane 4: #7<br />
*Lane 5: #8<br />
*Lane 6: -<br />
*Lane 7: 1 kb ladder<br />
*Lane 8: -<br />
<br />
*Refer to Taz Bam Eco Digest 8/18.tif for images<br />
*Digests 1,2,3,6,7,8, came out nicely<br />
<br />
*Gel extraction:<br />
**Mass (g), Mass+ gel (g), Gel (g), QG buffer:<br />
***(A) Tube 1-2: 1.0201, 1.1442, 0.1241, 372.3<br />
***(B) Tube 3: 1.0106, 1.0767, 0.0661, 198.3<br />
***(C) Tube 6-8: 1.0213, 1.2399, 0.2186, 655.8<br />
<br />
*Nanodrop concentrations: ng/μL<br />
**A: 8.9<br />
**B: 8.7<br />
**C: 15.9<br />
<br />
3) Transformation of Taz BB pGEM<br />
4) Stratagene<br />
<br />
<br />
August 19, 09<br />
<br />
*Ligation of TazBB-pGEM (10:30 am -10:45 pm)<br />
<br />
1) 6.19 μL H2O<br />
2) 1 μL 10x buffer<br />
3) 1 μL pGEM<br />
4) 1.31 μL Taz<br />
5) 0.5 μL DNA ligase<br />
<br />
<br />
*Transformations into RU 1012<br />
<br />
1) Taz pNoTat (4)+C<br />
2) TazBBpGEM (4)+C<br />
<br />
<br />
August 20, 09<br />
<br />
*Liquid culture: Testing RU1012 (Kanr) with Taz 1 (Ampr) <br />
*Negative: no growth<br />
*Amp: no growth<br />
*Amp+Kan: growth<br />
*Kan: growth<br />
*Chloremphenicol: no growth<br />
*A+K+C: no growth<br />
<br />
<br />
<br />
<br />
Week 10<br />
----<br />
<br />
1. 8/24/09: Received successful sequencing for Taz1 (p-GEM). <br />
2. Received GINKGO ompC-RFP (Tet): <br />
<br />
*Incubated plates, inoculated cultures, mini-prepped DNA <br />
<br />
3. Constructed Taz1 BB: <br />
*Digest (EcoR1, Pst1) and gel extraction of mini-prepped Taz1 BB (p-GEM)<br />
<br />
*Taz1 BB (p-GEM) Mini-Preps (Nanodrop Concentrations): Sample: Concentration, 260/280<br />
**1: 286.0, 1.93<br />
**2: 127.3, 1.98<br />
**3: 330.1, 1.92<br />
**4: 292.3, 1.95<br />
**5: 99.6, 1.98<br />
**6: 415.1, 1.92<br />
**7: 110.5, 1.94 <br />
<br />
<br />
Gel: Taz1 BB (p-GEM): <br />
<br />
<br />
<br />
*Gel Extraction Concentration: Taz1 BB (p-GEM): Sample: ng/uL, 260/280 <br />
**1: 9.8, 1.48 <br />
**2: 13.7, 1.78 <br />
<br />
*Digest (EcoR1, Pst1) and gel extraction of BB vector. <br />
<br />
Gel: BB Vector: <br />
<br />
<br />
<br />
<br />
<br />
<br />
*Gel Extraction Concentration: BB Vector <br />
**18.1 ng/uL, 260/280 = 1.83 <br />
<br />
*Overnight ligation of Taz1 BB and BB vector. <br />
<br />
*For 50 ng BB vector (2.78 uL of 18.1 ng/uL BB vector Gel Extract) (1458/2079) (3 or 6) = 105.19ng (10.73 uL of 9.8ng/uL Taz1 BB (p-GEM) Sample 1 Gel Extract) or 210.39 ng (21.47uL) Taz1 BB. <br />
<br />
*Transformation of ligation into DH5α. Incubated plates, inoculated liquid cultures, mini-prepped DNA, received successful sequencing.<br />
<br />
4. Stratagene Mutagenesis: <br />
<br />
(i) Thaw dNTP mix once; prepare single-use aliquots (-20°C)<br />
(ii) Control Reaction<br />
<br />
*5 μL 10 x rxn buffer; 2 μL pwhitescript control plasmid (4.5 kb); 1.25 μL control primers (1), (2); 1 μL dNTP mix; 38.5 μL ddH2O<br />
*After: 1 μL pFuUltra DNA polymerase<br />
<br />
*Reaction:<br />
**5 μL 20x buffer; DNA template (Taz pNoTat); primer (1 mut), primer (2 mut); 1 μL dNTP; X μL ddH2O<br />
**After: 1 μL PfuUltra DNA polymerase<br />
<br />
*Transformation→ plate: no colonies for both control snad samples<br />
<br />
5. Tested Sequencing of Taz1 (p-NOTat ): <br />
<br />
*Digest (BamH1, EcoR1) and received successful sequencing.<br />
<br />
*Taz1 (p-NOTat) Mini-Preps (Nanodrop Concentrations): Sample: Concentration, 260/280 <br />
**1: 178.4, 1.97 <br />
**2: 188.6, 1.96<br />
**3: 168.7, 1.96<br />
**4: 371.2, 1.91<br />
**5: 197.2, 1.95<br />
**6: 137.5, 2.00<br />
**7: 198.4, 1.97 <br />
<br />
<br />
<br />
Week 12<br />
----<br />
<br />
September 10, 09<br />
<br />
(i) control: <br />
*5 μL 10x reaction buffer<br />
*2 μL pwhitescript<br />
*1.25 μL control primer 1<br />
*1.25 μL control primer 2<br />
*1 μL dNTP<br />
*38.5 μL ddH2O<br />
**then 1 μL PfuUltra HF DNA polymerase<br />
<br />
(ii) sample (same except 2 μL pNoTat template 6 (137.5 ng/μL)<br />
<br />
*Result: no colonies<br />
<br />
<br />
September 13, 09<br />
<br />
*Transformation of RU1012<br />
*T1: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)<br />
*T2: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)<br />
*T3: RU1012+ OmpC-RFP + Taz→ Tet-Amp plate (3:50 pm)<br />
*T4: RU1012+ OmpC-RFP+ Taz→ Tet=Amp Plate (3:50 pm)<br />
*T5: RU1012→ Tet plate (3:20 pm)<br />
<br />
<br />
<br />
<br />
Week 13<br />
----<br />
<br />
<br />
September 14, 09, 7 am<br />
<br />
*Check plates: RU1012 negative control→ lots of growth!!! Batch of plates is bad.<br />
**(200 μL) RU1012+OmpC-RFP→ lots of growth<br />
**(50 μL) RU1012+ OmpC-RFP→ lots of growth<br />
**MC IPTG+ Xgal→ no growth<br />
**Sample 1 250 μL → no growth<br />
**Sample 2 250 μL→ 3 colonies<br />
**TC IPTG+ X-gal→ 6-7 colonies<br />
**RU1012+Taz+Ompc-RFP 1→ no growth<br />
**RU1012+ Taz+ OmpC-RFP 2→ growth<br />
<br />
September 15 -16, 09<br />
<br />
*Testing Aspartate binding of Double transformants<br />
*8:15 pm: start 10 liquid cultures in minimal media with Amp, Tet, Kan<br />
*10:15 am: move 0.5 mL to new 5 mL culture tube (minimal media+ A, T,K)<br />
<br />
*Liquid cultures didn’t grow<br />
*Redid cultures→ 16 of them <br />
<br />
(i) LB→ growth<br />
(ii) LB+A→ growth<br />
(iii) LB+T→growth<br />
(iv) LB+A+T→ no growth<br />
(v) MM→ no growth<br />
(vi) MM+A→ no growth<br />
(vii) MM+T→ no growth<br />
(viii) MM+A+T→ no growth<br />
<br />
*Plates (LB amp, tet-new plates)<br />
*IPTG induced: re-streak<br />
<br />
*IPTG induced RU1012 Double transformation<br />
**OmpC-RFP (Tetr)<br />
**Taz pNotat miniprep1 from 8/21/09(Ampr)<br />
<br />
*Redid double transformation of RU1012 with Taz-pNoTat+OmpC-RFP<br />
*2:30 pm: visualized under fluroscope→ WE SEE RED!! DOUBLE TRANSFORMATION SUCCESS.<br />
<br />
<br />
<br />
*Liquid cultures (picked only red colonies) 5 μL<br />
**LB (negative)→ growth<br />
**LB→ growth<br />
**LB+A→ growth<br />
**LB+T→ no growth<br />
**LB+A+T→ no growth<br />
**MM (neg)→ no growth<br />
**MM→ no growth<br />
**MM+A→ no growth<br />
**MM+T→ no growth<br />
**MM+A+T→ no growth<br />
<br />
September 18, 09<br />
<br />
*To do: try liquid cultures with less Tet<br />
*Single transformation of RFP (TetR) Construct→RU1012+ DH5α<br />
*Plate on only Tet plate (make sure to use the TetR construct is usd and not the first KanR construct we received)<br />
<br />
<br />
<br />
Week 14<br />
----<br />
<br />
<br />
<br />
September 21, 09<br />
<br />
*Retry liquid cultures from LB-A-T plate<br />
<br />
*Amp Tet Growth<br />
*- - Yes<br />
*- 0.1 μL Yes<br />
*- 0.5 μL Yes<br />
*- 1 μL Yes<br />
*- 2 μL Yes<br />
*- 4 μL Yes<br />
*5 μL 0.1 μL Yes<br />
*5 μL 0.5 μL Yes<br />
*5 μL 1 μL Yes<br />
*5 μL 2 μL Yes<br />
*5 μL 4 μL Yes<br />
<br />
*IPTG induction (5 μL) at 10:25 am<br />
**0.5 mL liquid culture<br />
**5 mL Minimal media<br />
**5 μL IPTG<br />
<br />
*Result: All fluoresced red…there is lactose in LB.<br />
<br />
<br />
September 23, 09<br />
<br />
*Minimal media liquid cultures (1pm→ 10 am)<br />
**MM (neg)→ no growth<br />
**MM (pos)+ RU1012 with Taz 1 from 8/20→ growth<br />
**MM with double transformants→ growth<br />
**MM+A with double transformants→ no growth<br />
**MM+T with double transformants→ no growth<br />
**MM+A+T with double transformants→ no growth<br />
<br />
*Testing for RFP (5mL cultures)<br />
1) no IPTG no aspartate<br />
2) no IPTG 100 μL 0.1M Asp (2mM)<br />
3) 5 μL IPTG 50 μL Asp (1mM)<br />
4) 5 μL IPTG 100 μL Asp (2mM)<br />
5) 5 μL IPTG 10 μL Asp (0.2 mM)<br />
6) 5 μL IPTG (3 hours later)→ 10 μL Asp<br />
7) 5 μL IPTG (3 hours later)→ 50 μL Asp<br />
8) 5 μL IPTG (3 hours later)→ 100 μL Asp<br />
<br />
*0.1 M Aspartate solution: 1.33 g in 100mL dH2O</div>Ashleyhttp://2009.igem.org/File:Polyala_rpb_img.pngFile:Polyala rpb img.png2009-10-22T00:32:56Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-22T00:23:22Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
<br />
'''<big>Re-engineering Chemoreceptor #1: Ribose Binding Protein</big>'''<br />
<br />
[[Image:Rbp img.png|300px]]<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on 100 of Brown's Center for Computational Molecular Biology (http://www.brown.edu/Research/CCMB/) clustered servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. The scores for the top few final mRBP designs (the selected design highlighted in bold):<br />
<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500. Several of the designed proteins scored as good as or better than the natural RBP in the relevant categories (see tables below). The final design (in bold) selected had the best overall scores in each category.<br />
<br />
[[Image:Final_sel.png|800px|center|thumb|The final selection list of mRBP.]]<br />
[[Image:native_score.png|800px|center|thumb|The score of the native RBP]]<br />
<br />
{|<br />
!total_score<br />
!tot_pstat_pm<br />
!tot_nlpstat_pm<br />
!tot_burunsat_pm<br />
!tot_hbond<br />
!SR_1_interf_E_1_2<br />
!SR_1_dasa_1_2<br />
|-<br />
|Weighted sum of the other terms. (Lower better)<br />
|How well packed the protein is. (0-1)<br />
|How well packed the protein is without the ligand. (0-1)<br />
|Number buried unsatisfied polars. (Lower better)<br />
|Number of H-bonds. (Higher better)<br />
|How well the ligand binds to protein. (Lower better)<br />
|How exposed the ligand is. (0-1)<br />
|}<br />
<br />
<br />
[[Image:MRBP.png|400px| thumb|The mutated Ribose Binding Protein]][[Image:ligandbindingprotein.png|400px|thumb|Histamine in the mutated ligand binding pocket]] <br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode, run on the same cluster. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
<br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''<big>Re-engineering Chemoreceptor #2: Tar Receptor</big>'''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
In normal E. coli cells, aspartate binds to the Tar receptor. The Tar-EnvZ chimera protein (created by Utsumi et. al in "Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate" 1989) allowed the Taz protein to be linked to the EnvZ cascade in the same manner as Trg-EnvZ. Just as in that system, ligand binding to its receptor leads to an intracellular signaling cascade promoting gene transcription. <br />
<br />
'''<big>Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription</big>'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). '''AR-just put a link here instead of saying it''' The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
All photographs were taken on an epi-fluorescent microscope: Olympus SZX16, excitation source X-cite Series 120. The first image in each series is under bright light, the second under a fluorescent filter for RFP. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]<br />
<br />
<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_HBP"><br />
<img src="https://static.igem.org/mediawiki/2009/d/d8/Brown_hbp_bottom_3.png"><br />
</html></div>Ashleyhttp://2009.igem.org/File:Rbp_img.pngFile:Rbp img.png2009-10-22T00:21:54Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-22T00:17:57Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
<br />
'''<big>Re-engineering Chemoreceptor #1: Ribose Binding Protein</big>'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on 100 of Brown's Center for Computational Molecular Biology (http://www.brown.edu/Research/CCMB/) clustered servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. The scores for the top few final mRBP designs (the selected design highlighted in bold):<br />
<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500. Several of the designed proteins scored as good as or better than the natural RBP in the relevant categories (see tables below). The final design (in bold) selected had the best overall scores in each category.<br />
<br />
[[Image:Final_sel.png|800px|center|thumb|The final selection list of mRBP.]]<br />
[[Image:native_score.png|800px|center|thumb|The score of the native RBP]]<br />
<br />
{|<br />
!total_score<br />
!tot_pstat_pm<br />
!tot_nlpstat_pm<br />
!tot_burunsat_pm<br />
!tot_hbond<br />
!SR_1_interf_E_1_2<br />
!SR_1_dasa_1_2<br />
|-<br />
|Weighted sum of the other terms. (Lower better)<br />
|How well packed the protein is. (0-1)<br />
|How well packed the protein is without the ligand. (0-1)<br />
|Number buried unsatisfied polars. (Lower better)<br />
|Number of H-bonds. (Higher better)<br />
|How well the ligand binds to protein. (Lower better)<br />
|How exposed the ligand is. (0-1)<br />
|}<br />
<br />
<br />
[[Image:MRBP.png|400px| thumb|The mutated Ribose Binding Protein]][[Image:ligandbindingprotein.png|400px|thumb|Histamine in the mutated ligand binding pocket]] <br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode, run on the same cluster. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
<br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''<big>Re-engineering Chemoreceptor #2: Tar Receptor</big>'''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
In normal E. coli cells, aspartate binds to the Tar receptor. The Tar-EnvZ chimera protein (created by Utsumi et. al in "Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate" 1989) allowed the Taz protein to be linked to the EnvZ cascade in the same manner as Trg-EnvZ. Just as in that system, ligand binding to its receptor leads to an intracellular signaling cascade promoting gene transcription. <br />
<br />
'''<big>Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription</big>'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). '''AR-just put a link here instead of saying it''' The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
All photographs were taken on an epi-fluorescent microscope: Olympus SZX16, excitation source X-cite Series 120. The first image in each series is under bright light, the second under a fluorescent filter for RFP. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]<br />
<br />
<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_HBP"><br />
<img src="https://static.igem.org/mediawiki/2009/d/d8/Brown_hbp_bottom_3.png"><br />
</html></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T23:51:13Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
<br />
'''<big>Re-engineering Chemoreceptor #1: Ribose Binding Protein</big>'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
'''AR-Need pictures!! Throw in pic of RBP and ribose. Then have an ala mutated pocket. Then have a pic of histamine in the naked pocket and finally one of the top choices with histamine in the pocket. Use the same angle and cutaway of the pocket in every case.'''<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on 100 of Brown's Center for Computational Molecular Biology (http://www.brown.edu/Research/CCMB/) clustered servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. The scores for the top few final mRBP designs (the selected design highlighted in bold):<br />
[[File:Final_sel.png]]<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500. The relevant scores for the top mRBP designs (the one synthesized in bold):<br />
[[File:final_sel.tiff|mRBP Scores]]<br />
<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode, run on the same cluster. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
[[Image:ligandbindingprotein.png|400px|thumb|The mutated ligand binding pocket for histamine]] [[Image:MRBP.png|400px| thumb|The mutated Ribose Binding Protein]]<br />
<br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
'''<big>Re-engineering Chemoreceptor #2: Tar Receptor</big>'''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
In normal E. coli cells, aspartate binds to the Tar receptor. The Tar-EnvZ chimera protein (created by Utsumi et. al in "Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate") allowed the Taz protein to be linked to the EnvZ cascade in the same manner as Trg-EnvZ. Just as in that system, ligand binding to its receptor leads to an intracellular signaling cascade promoting gene transcription. <br />
<br />
'''<big>Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription</big>'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering [https://2009.igem.org/Team:Brown/Project_HBP gene transcription]. The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
All photographs were taken on an epi-fluorescent microscope: Olympus SZX16, excitation source X-cite Series 120. The first image in each series is under bright light, the second under a fluorescent filter for RFP. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]<br />
<br />
<br />
<br />
<html><br />
<a href="https://2009.igem.org/Team:Brown/Project_HBP"><br />
<img src="https://static.igem.org/mediawiki/2009/d/d8/Brown_hbp_bottom_3.png"><br />
</html></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-21T23:16:56Z<p>Ashley: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''<big>The Allergic Response</big>'''<br />
----<br />
<br />
The prevalence of food, seasonal, and other allergies has been rapidly increasing in recent times. In particular, over 50 million people in the United States suffer from allergic rhinitis, more commonly known as hay fever. This allergy is caused by allergens such as pollen or dust and causes the mucous membranes of the eyes and nose to become itchy and inflamed, resulting in irritating symptoms such as runny nose and watery eyes. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
<br />
Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these receptors. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods which often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
<br />
<br />
<Video coming soon!></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-21T23:16:41Z<p>Ashley: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
'''<big>The Allergic Response</big>'''<br />
----<br />
<br />
The prevalence of food, seasonal, and other allergies has been rapidly increasing in recent times. In particular, over 50 million people in the United States suffer from allergic rhinitis, more commonly known as hay fever. This allergy is caused by allergens such as pollen or dust and causes the mucous membranes of the eyes and nose to become itchy and inflamed, resulting in irritating symptoms such as runny nose and watery eyes. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
<br />
Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these receptors. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods which often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
<br />
<br />
<Video coming soon!></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_IntroductionTeam:Brown/Project Introduction2009-10-21T23:16:17Z<p>Ashley: /* Introduction */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''<big>The Allergic Response</big>'''<br />
----<br />
<br />
The prevalence of food, seasonal, and other allergies has been rapidly increasing in recent times. In particular, over 50 million people in the United States suffer from allergic rhinitis, more commonly known as hay fever. This allergy is caused by allergens such as pollen or dust and causes the mucous membranes of the eyes and nose to become itchy and inflamed, resulting in irritating symptoms such as runny nose and watery eyes. Histamine has been identified as a principal mediator of inflammatory responses. Upon first contact with an allergen, plasma cells release Immunoglobulin E (IgE) antibodies. These antibodies then activate mast cell degranulation and release of histamine. When histamine reaches histamine receptors on various target cells, vasodilation and inflammation occurs, resulting in allergic symptoms. <br />
<br />
Allergies are routinely treated with antihistamine drugs, which have many adverse effects. Antihistamines compete with histamine to bind and block these receptors. For many people, sedation remains the primary concern when considering the adverse effects of the newer antihistamines, particularly since these drugs are given to patients with chronic disorders with treatment periods which often extend over several months or even years. Besides sedation, there also exists concern regarding the caridotoxicity of antihistamines and other adverse drug interactions. For patients suffering from chronic allergies and inflammation, there is a great need for an alternative strategy for combating allergic symptoms without causing significant side effects.<br />
<br />
<br />
<Video coming soon!></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T22:04:39Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
'''AR-Need pictures!! Throw in pic of RBP and ribose. Then have an ala mutated pocket. Then have a pic of histamine in the naked pocket and finally one of the top choices with histamine in the pocket. Use the same angle and cutaway of the pocket in every case.'''<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. '''AR-throw in a link to the computer cluster at Brown'''<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
'''AR-put in part of Excel spreadsheet with all the sortable criteria, just show top 10 or whatever...'''<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). '''AR-just put a link here instead of saying it''' The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
All photographs were taken on an epi-fluorescent microscope: Olympus SZX16, excitation source X-cite Series 120. The first image in each series is under bright light, the second under a fluorescent filter for RFP. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T22:03:41Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
'''AR-Need pictures!! Throw in pic of RBP and ribose. Then have an ala mutated pocket. Then have a pic of histamine in the naked pocket and finally one of the top choices with histamine in the pocket. Use the same angle and cutaway of the pocket in every case.'''<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. '''AR-throw in a link to the computer cluster at Brown'''<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
'''AR-put in part of Excel spreadsheet with all the sortable criteria, just show top 10 or whatever...'''<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). '''AR-just put a link here instead of saying it''' The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
All photographs were taken on an epi-fluorescent microscope: Olympus SZX16, excitation source X-cite Series 120. The first image in each series is under bright light, the second under a fluorescent filter for RFP. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T21:57:54Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
'''AR-Need pictures!! Throw in pic of RBP and ribose. Then have an ala mutated pocket. Then have a pic of histamine in the naked pocket and finally one of the top choices with histamine in the pocket. Use the same angle and cutaway of the pocket in every case.'''<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. '''AR-throw in a link to the computer cluster at Brown'''<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
'''AR-put in part of Excel spreadsheet with all the sortable criteria, just show top 10 or whatever...'''<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). '''AR-just put a link here instead of saying it''' The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
'''AR-show pictures of data'''<br />
<br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg|300px]] [[Image:RU1012 Taz RFP.jpg|300px]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T21:56:18Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
'''AR-Need pictures!! Throw in pic of RBP and ribose. Then have an ala mutated pocket. Then have a pic of histamine in the naked pocket and finally one of the top choices with histamine in the pocket. Use the same angle and cutaway of the pocket in every case.'''<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. '''AR-throw in a link to the computer cluster at Brown'''<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
'''AR-put in part of Excel spreadsheet with all the sortable criteria, just show top 10 or whatever...'''<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). '''AR-just put a link here instead of saying it''' The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
'''AR-show pictures of data'''<br />
<br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg|300px]] [[Image:RU1012 RFP.jpg|300px]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg|300px]] [[Image:RU1012 OmpC RFP.jpg|300px]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg|300px]] [[Image:Image:RU1012 Taz RFP.jpg|300px]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg|300px]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg|300px]] [[Image:RU1012 Taz OmpC RFP.jpg|300px]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg|300px]] [[Image:RU1012 Trg OmpC RFP.jpg|300px]]</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_All_TogetherTeam:Brown/Project All Together2009-10-21T21:50:16Z<p>Ashley: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
[[Image:Cascade.gif]]<br />
<br />
<br />
1) Mast cells release histamine during the allergic response.<br />
<br />
2) Histamine binds to our re-engineered histamine receptor.<br />
<br />
3) This receptor’s intracellular kinase domain EnvZ phosphorylates transcription factor OmpR.<br />
<br />
4) OmpR turns on transcription of DNA under the OmpC promoter.<br />
<br />
5) The genes for rEV131 and its attached secretion signal are transcribed.<br />
<br />
6) After translation, the secretion peptide allows rEV131 to be released into the extracellular fluid.<br />
<br />
7) rEV131 sequesters histamine<br />
<br />
8) The transcription and secretion of rEV131 continues as long as histamine is present in the extracellular fluid. When histamine concentration returns to its pre-allergic response state, production of rEV131 stops because the initiating ligand histamine is no longer present.</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_All_TogetherTeam:Brown/Project All Together2009-10-21T21:49:13Z<p>Ashley: </p>
<hr />
<div>{{Brown}}<br />
[[Image:Cascade.gif]]<br />
<br />
<br />
1) Mast cells release histamine during the allergic response.<br />
<br />
2) Histamine binds to our re-engineered histamine receptor.<br />
<br />
3) This receptor’s intracellular kinase domain EnvZ phosphorylates transcription factor OmpR.<br />
<br />
4) OmpR turns on transcription of DNA under the OmpC promoter.<br />
<br />
5) The genes for rEV131 and its attached secretion signal are transcribed.<br />
<br />
6) After translation, the secretion peptide allows rEV131 to be released into the extracellular fluid.<br />
<br />
7) rEV131 sequesters histamine<br />
<br />
8) The transcription and secretion of rEV131 continues as long as histamine is present in the extracellular fluid. When histamine concentration returns to its pre-allergic response state, production of rEV131 stops because the initiating ligand histamine is no longer present.</div>Ashleyhttp://2009.igem.org/File:Cascade.gifFile:Cascade.gif2009-10-21T21:47:26Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T21:42:49Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
'''AR-Need pictures!! Throw in pic of RBP and ribose. Then have an ala mutated pocket. Then have a pic of histamine in the naked pocket and finally one of the top choices with histamine in the pocket. Use the same angle and cutaway of the pocket in every case.'''<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. '''AR-throw in a link to the computer cluster at Brown'''<br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
'''AR-put in part of Excel spreadsheet with all the sortable criteria, just show top 10 or whatever...'''<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. '''AR-throw in Figure from paper that shows this'''<br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). '''AR-just put a link here instead of saying it''' The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
'''AR-show pictures of data'''<br />
<br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
[[Image:RU1012 BL.jpg]] [[Image:RU1012 RFP.jpg]]<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
[[Image:RU1012 OmpC BL.jpg]] [[Image:RU1012 OmpC RFP.jpg]]<br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz BL.jpg]] [[Image:Image:RU1012 Taz RFP.jpg]]<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg RFP.jpg]]<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
[[Image:RU1012 Taz OmpC BL.jpg]] [[Image:RU1012 Taz OmpC RFP.jpg]]<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
[[Image:RU1012 Trg OmpC BL.jpg]] [[Image:RU1012 Trg OmpC RFP.jpg]]</div>Ashleyhttp://2009.igem.org/File:RU1012_Trg_OmpC_RFP.jpgFile:RU1012 Trg OmpC RFP.jpg2009-10-21T21:32:51Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_Trg_OmpC_BL.jpgFile:RU1012 Trg OmpC BL.jpg2009-10-21T21:31:57Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_Taz_OmpC_RFP.jpgFile:RU1012 Taz OmpC RFP.jpg2009-10-21T21:31:11Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_Taz_OmpC_BL.jpgFile:RU1012 Taz OmpC BL.jpg2009-10-21T21:30:13Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_Trg_RFP.jpgFile:RU1012 Trg RFP.jpg2009-10-21T21:26:18Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_Taz_RFP.jpgFile:RU1012 Taz RFP.jpg2009-10-21T21:24:25Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_Taz_BL.jpgFile:RU1012 Taz BL.jpg2009-10-21T21:23:57Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_RFP.jpgFile:RU1012 RFP.jpg2009-10-21T21:22:51Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_OmpC_RFP.jpgFile:RU1012 OmpC RFP.jpg2009-10-21T21:22:12Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_OmpC_BL.jpgFile:RU1012 OmpC BL.jpg2009-10-21T21:21:42Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/File:RU1012_BL.jpgFile:RU1012 BL.jpg2009-10-21T21:18:58Z<p>Ashley: </p>
<hr />
<div></div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T21:01:28Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. <br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. <br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response when allergens are present.<br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription under the OmpC promoter, we have constructed a cassette that places the OmpC promoter over the RFP gene for red fluorescence. Testing for functionality of the cascade then simply involved observing whether RFP expression occurred under ompC, a simple fluorescence assay conducted on an epifluorescence microscope. Qualitative visualization of red-fluorescing colonies transformed with both the receptor and cascade components indicated a functional intracellular signaling system. <br />
<br />
We have tested this signaling cascade by performing the following series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. As efforts to construct a novel histamine receptor were being conducted in parallel to these assays, testing of the cascade was conducted with the original chimeric chemoreceptor Tar-EnvZ. The binding of the wild-type ligand aspartate to Tar and the intracellular transcription of ompC-RFP it initiated was thus used as a proof-of-concept of our future histamine-initiated system. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ<br />
<br />
'''Results: Testing the Cascade'''<br />
<br />
Our fluorescence assays of these transformed colonies indicate that signal transduction is indeed effective. <br />
<br />
1. RU1012 with no plasmid<br />
<br />
2. RU1012 with OmpC-RFP <br />
<br />
3. RU1012 with Tar-EnvZ<br />
<br />
4. RU1012 with Trg-EnvZ<br />
<br />
5. RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
6. RU1012 with OmpC-RFP + Trg-EnvZ</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T20:32:40Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. <br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. <br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the functional receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR thereby activating gene transcription under the ompC promoter. By replacing the gene normally present under this promoter with our gene of interest, we successfully manipulated the cascade to produce an appropriate cellular response to the release of allergens. <br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription of DNA under the OmpC promoter, we have constructed a cassette from the registry that places the OmpC promoter over the gene for RFP. With this OmpC-RFP reporter cassette, we can test the functionality of the intracellular cascade. <br />
<br />
We have tested this signaling cascade by performing a series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. Results indicate that signal transduction is effective.<br />
<br />
RU1012 with no plasmid<br />
<br />
RU1012 with OmpC-RFP <br />
<br />
RU1012 with Tar-EnvZ<br />
<br />
RU1012 with Trg-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Trg-EnvZ</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T19:59:28Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. <br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. <br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). Chimeric proteins were created by fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ. Activation of the receptor domains by histamine binding is thus able to initiate an intracellular cascade that phosphorylates the transcription factor ompR and activates gene transcription under the ompC promoter. By replacing the gene <br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription of DNA under the OmpC promoter, we have constructed a cassette from the registry that places the OmpC promoter over the gene for RFP. With this OmpC-RFP reporter cassette, we can test the functionality of the intracellular cascade. <br />
<br />
We have tested this signaling cascade by performing a series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. Results indicate that signal transduction is effective.<br />
<br />
RU1012 with no plasmid<br />
<br />
RU1012 with OmpC-RFP <br />
<br />
RU1012 with Tar-EnvZ<br />
<br />
RU1012 with Trg-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Trg-EnvZ</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T19:54:05Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. <br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. <br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). The successful mutation of the binding pockets on both RBP and Tar was therefore followed by the complete construction of each receptor as it would function in the cell membrane. For both RBP and Tar, this meant replicating the work done by Masayori Inouye et. al to create the chimeric proteins Trg-EnvZ (Trz) and Tar-EnvZ (Taz) (respectively). By fusing the intracellular domains of Tar and Trg (Trg is the membrane receptor associated with ligand-bound RBP) to the kinase domain of EnvZ, activation of the receptor domains by histamine binding initiates an intracellular cascade that phosphorylates the transcription factor ompR, activating the transcription of the gene of interest located under the ompC promoter. <br />
<br />
In normal E. coli cells, binding of Aspartate to Tar initiates an intracellular cascade that signals chemotaxis. Dr. Masayori Inouye et al, using the same methodology as that which created the Trg-EnvZ chimera, fused the intracellular domain of Tar to the kinase domain of EnvZ. Thus, activation of the Tar receptor domain will cause its EnvZ domain to phosphorylate the transcription factor OmpR, which will subsequently activate the transcription of DNA under the OmpC promoter. <br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription of DNA under the OmpC promoter, we have constructed a cassette from the registry that places the OmpC promoter over the gene for RFP. With this OmpC-RFP reporter cassette, we can test the functionality of the intracellular cascade. <br />
<br />
We have tested this signaling cascade by performing a series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. Results indicate that signal transduction is effective.<br />
<br />
RU1012 with no plasmid<br />
<br />
RU1012 with OmpC-RFP <br />
<br />
RU1012 with Tar-EnvZ<br />
<br />
RU1012 with Trg-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Trg-EnvZ</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T18:55:05Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. <br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. <br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
After successful mutation of both receptors (RBP and Tar) <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). <br />
<br />
In normal E. coli cells, binding of Aspartate to Tar initiates an intracellular cascade that signals chemotaxis. Dr. Masayori Inouye et al, using the same methodology as that which created the Trg-EnvZ chimera, fused the intracellular domain of Tar to the kinase domain of EnvZ. Thus, activation of the Tar receptor domain will cause its EnvZ domain to phosphorylate the transcription factor OmpR, which will subsequently activate the transcription of DNA under the OmpC promoter. <br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription of DNA under the OmpC promoter, we have constructed a cassette from the registry that places the OmpC promoter over the gene for RFP. With this OmpC-RFP reporter cassette, we can test the functionality of the intracellular cascade. <br />
<br />
We have tested this signaling cascade by performing a series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. Results indicate that signal transduction is effective.<br />
<br />
RU1012 with no plasmid<br />
<br />
RU1012 with OmpC-RFP <br />
<br />
RU1012 with Tar-EnvZ<br />
<br />
RU1012 with Trg-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Trg-EnvZ</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T18:52:03Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. <br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. <br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
The creation of a novel histamine receptor to detect elevated histamine is only the first step to providing an appropriate cell response to allergens. Once a functional histamine sensor is inserted into the cell membrane, its activation must be linked to an intracellular cascade responsible for triggering gene transcription (See "Histamine Binding Protein" for more information on the particular gene engineered to deal with the allergic response). <br />
<br />
In normal E. coli cells, binding of Aspartate to Tar initiates an intracellular cascade that signals chemotaxis. Dr. Masayori Inouye et al, using the same methodology as that which created the Trg-EnvZ chimera, fused the intracellular domain of Tar to the kinase domain of EnvZ. Thus, activation of the Tar receptor domain will cause its EnvZ domain to phosphorylate the transcription factor OmpR, which will subsequently activate the transcription of DNA under the OmpC promoter. <br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription of DNA under the OmpC promoter, we have constructed a cassette from the registry that places the OmpC promoter over the gene for RFP. With this OmpC-RFP reporter cassette, we can test the functionality of the intracellular cascade. <br />
<br />
We have tested this signaling cascade by performing a series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. Results indicate that signal transduction is effective.<br />
<br />
RU1012 with no plasmid<br />
<br />
RU1012 with OmpC-RFP <br />
<br />
RU1012 with Tar-EnvZ<br />
<br />
RU1012 with Trg-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Trg-EnvZ</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_Histamine_SensorTeam:Brown/Project Histamine Sensor2009-10-21T18:30:12Z<p>Ashley: /* Histamine Sensor */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Histamine Sensor=<br />
<br />
<br />
<br />
During the allergic response, the concentration of histamine in the extracellular fluid of the nasal cavity increases. To initiate a response; therefore, a histamine sensor is necessary.<br />
<br />
Because natural histamine receptors exist only in eukaryotic cells as G-coupled protein receptors, they are unusable for our prokaryotic system. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular cascade, signaling cells to respond appropriately to the increase in histamine concentration. <br />
<br />
To engineer a novel histamine receptor, we are mutating two existing prokaryotic chemoreceptors so that they bind histamine rather than their wild type ligands. <br />
<br />
'''Re-engineering Chemoreceptor #1: Ribose Binding Protein'''<br />
<br />
We modified ribose binding protein (RBP), which normally binds ribose in the periplasmic space of Escherichia coli, to bind histamine. Our computational approach to accomplish this task was modeled after that taken in Looger et al., "Computational design of receptor and sensor proteins with novel functions" (2003). Using Rosetta macromolecular modeling software, we modified the program's existing Enzyme Design Function (such as that used in Röthlisberger et al "Kemp elimination catalysts by computational enzyme design" (2008)) to enable the re-design of RBP, a non-enzymatic protein. The successful modification of RBP would result in its ability to bind histamine. <br />
<br />
How we designed the protein:<br />
<br />
1) Took the PDB file for the crystal structure of RBP cocrystallized with ribose (2DRI). Removed water molecules and added missing hydrogens. <br />
<br />
2) Used UCSF Chimera to geometrically search for all van der Waals interactions between ribose and RBP in the crystal structure. Identified the amino acids responsible for these interactions as those most likely present in the ligand binding pocket of RBP. <br />
<br />
3) Used UCSF Chimera to mutate all the identified residues in RBP to alanine (which has neutral chemical properties, and almost no side chains), thereby effectively creating a "blank" version of RBP, one that has no specific binding pocket for any ligand (removing its binding affinity for ribose).<br />
<br />
4) Used Rosetta's Ligand Docking mode on a cluster of 100 servers to replace ribose in the alanine-mutated RBP (polyala RBP) with a 3D structure for histamine. <br />
<br />
5) To isolate histamine's lowest energy conformation in RBP, used Monte Carlo minimization to find the relative orientations of both components that minimize steric contacts while still keeping histamine roughly within the original ligand binding pocket. Generated 10000 PDB files of histamine docked to the polyala RBP.<br />
<br />
6) Sorted the 10000 docked PDB files by their interface energies between ligand and protein. Selected the top 2500.<br />
<br />
7) Took the top 2500 and input them into the Rosetta Enzyme Design mode. Specified the residues mutated to alanine as those to re-design. Used Rosetta Design’s probabilistic simulated annealing algorithm to find the particular residues that minimize total energy between protein and ligand (minimized energy indicates a stable state, favoring binding). Final designs are not guaranteed to yield the lowest possible energy conformations. However, by doing thousands of designs in parallel, we increased the likelihood of isolating mutations that result in histamine binding. <br />
<br />
8) Sorted through the output designs using several criteria: predicted interface energies between the protein and histamine, amount of hydrogen bonding between the protein and histamine (H-bonds are very good for ligand binding), and predicted folding ability of protein. <br />
<br />
The DNA for the top design was synthesized by GeneArt AG. We are in the process of testing the protein’s ability to bind histamine. <br />
<br />
In normal E. coli cells, ribose binding to RBP forms a ribose-RBP complex that interacts with the periplasmic domain of Trg, which in turn activates an intracellular cascade that induces chemotaxis. <br />
To link activation of Trg to induction of gene expression, we created a fusion between Trg periplasmic receptor domain and the EnvZ intracellular kinase domain by stitching the two intracellular domains at a shared NdeI restriction site. (as done in Baumgartner JW, et al. “Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ” (1994)). EnvZ phosphorylates the transcription factor OmpR, which causes transcription of DNA regulated by OmpC promoter. <br />
<br />
'''Re-engineering Chemoreceptor #2: Tar Receptor '''<br />
<br />
In parallel, we modified the receptor Tar, which normally binds aspartate in the periplasmic space of E. coli, to bind histamine. <br />
<br />
Loren Looger at the Howard Hughes Medical Institute's Janelia Farm campus used his protein design software Chameleon to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round-the-Horn Site-Directed Mutagenesis” protocol on OpenWetWare and Strategene’s Quikchange Mutagenesis II Kit. <br />
<br />
'''Initiating an Appropriate Response: Linking Histamine Sensation to Intracellular Transcription'''<br />
<br />
In normal E. coli cells, binding of Aspartate to Tar initiates an intracellular cascade that signals chemotaxis. Dr. Masayori Inouye et al, using the same methodology as that which created the Trg-EnvZ chimera, fused the intracellular domain of Tar to the kinase domain of EnvZ. Thus, activation of the Tar receptor domain will cause its EnvZ domain to phosphorylate the transcription factor OmpR, which will subsequently activate the transcription of DNA under the OmpC promoter. <br />
<br />
'''Assay to Test Histamine Sensitivity and Signaling Cascade Functionality'''<br />
<br />
Our assay to test these receptors’ affinities for histamine is based on fluorescence. Because the intracellular cascades of both Trg-EnvZ and Tar-EnvZ induce transcription of DNA under the OmpC promoter, we have constructed a cassette from the registry that places the OmpC promoter over the gene for RFP. With this OmpC-RFP reporter cassette, we can test the functionality of the intracellular cascade. <br />
<br />
We have tested this signaling cascade by performing a series of transformations. The E. coli strain RU1012 was used, because it is an EnvZ knockout strain. Results indicate that signal transduction is effective.<br />
<br />
RU1012 with no plasmid<br />
<br />
RU1012 with OmpC-RFP <br />
<br />
RU1012 with Tar-EnvZ<br />
<br />
RU1012 with Trg-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Tar-EnvZ<br />
<br />
RU1012 with OmpC-RFP + Trg-EnvZ</div>Ashleyhttp://2009.igem.org/Team:Brown/Links_AcknowledgementsTeam:Brown/Links Acknowledgements2009-10-21T17:50:28Z<p>Ashley: /* Acknowledgements */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Acknowledgements=<br />
<br />
<br />
<br />
'''We would like to thank the following individuals without whom this project would not have been made possible:'''<br />
<br />
*Dr. '''Gary Wessel''', for his invaluable advice, unremitting patience, continuous encouragement and most of all, tremendous enthusiasm for our project. <br />
<br />
*Graduate students '''Adrian Reich''', iGEM Adviser, and '''Diana Donovan''', for their continuous guidance and experimental support throughout the project. <br />
<br />
*'''John Szymanski''', iGEM alum, for his original competent cells protocol and generous assistance in the lab. <br />
<br />
<br />
<br />
*'''Adella Francis''', for her consistent and generous administrative assistance for Brown iGEM teams past and present.<br />
<br />
*'''John Cumbers''', Founder and continual supporter of Brown iGEM Teams.<br />
<br />
<br />
*The '''PRIMO Lab''', for their generous assistance in research protocols and laboratory facility use.<br />
<br />
*The '''Barnea Lab''', for the use of their laboratory facilities.<br />
<br />
*The Faculty Panel, for its invaluable support and advice.<br />
<br />
<br />
*The '''Brown UTRA Program''', for undergraduate summer research funding at Brown University. <br />
<br />
*Brown University Departments of Biology and Medicine, Engineering, Computational Biology, Molecular, Cell Biology, and Biochemistry.<br />
<br />
*'''Neil Parikh''', '''Kate Jacobs''', '''Rima Shah''', '''John Szymanski''' and '''Aaron Glieberman''': Former Brown iGEM Team Members who helped train and guide us, setting a high standard for all future iGEM Mentors. <br />
<br />
<br />
<br />
----<br />
<br />
<br />
*Dr. '''Guido Paesen''', Centre for Ecology & Hydrology, Oxford: for providing the initial inspiration to propose a project concerning the practical implications of a histamine binding protein, and also for generously sharing rEV131.<br />
<br />
*Dr. '''Loren Looger''', Howard Hughes Medical Institute: for the use of his computational protein design program to calculate mutations that would transform Tar’s aspartate binding pocket to one that selectively binds histamine. <br />
<br />
*Dr. '''Masayori Inouye''', Rutgers University: for his generous provision of Tar-EnvZ.<br />
<br />
*Dr. '''Luciano Marraffini''', Sontheimer Lab at Northwestern University, for the provision of plasmid pLM6, a shuttle vector between S.epidermidis and E.coli.<br />
<br />
*Dr. '''Reinholb Bruckner''', for the provision of shuttle vectors PRB474 and PRB473. <br />
<br />
<br />
----</div>Ashleyhttp://2009.igem.org/Team:Brown/Links_AcknowledgementsTeam:Brown/Links Acknowledgements2009-10-21T16:58:40Z<p>Ashley: /* Acknowledgements */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Acknowledgements=<br />
<br />
<br />
<br />
'''We would like to thank the following individuals without whom this project would not have been made possible:'''<br />
<br />
*Dr. '''Gary Wessel''', for his invaluable advice, unremitting patience, continuous encouragement and most of all, tremendous enthusiasm for our project. <br />
<br />
*Graduate students '''Adrian Reich''', iGEM Adviser, and '''Diana Donovan''', for their continuous guidance and experimental support throughout the project. <br />
<br />
*'''John Szymanski''', iGEM alum, for his original competent cells protocol and generous assistance in the lab. <br />
<br />
<br />
<br />
*'''Adella Francis''', for her consistent and generous administrative assistance for Brown iGEM teams past and present.<br />
<br />
*'''John Cumbers''', Founder and continual supporter of Brown iGEM Teams.<br />
<br />
<br />
*The '''PRIMO Lab''', for their generous assistance in research protocols and laboratory facility use.<br />
<br />
*The '''Barnea Lab''', for the use of their laboratory facilities.<br />
<br />
*The Faculty Panel, for its invaluable support and advice.<br />
<br />
<br />
*The '''Brown UTRA Program''', for undergraduate summer research funding at Brown University. <br />
<br />
*Brown University Departments of Biology and Medicine, Engineering, Computational Biology, Molecular, Cell Biology, and Biochemistry.<br />
<br />
*'''Neil Parikh''', '''Kate Jacobs''', '''Rima Shah''', '''John Szymanski''' and '''Aaron Glieberman''': Former Brown iGEM Team Members who helped train and guide us, setting a high standard for all future iGEM Mentors. <br />
<br />
<br />
<br />
----<br />
<br />
<br />
*Dr. '''Guido Paesen''', Centre for Ecology & Hydrology, Oxford: for providing the initial inspiration to propose a project concerning the practical implications of a histamine binding protein, and also for generously sharing rEV131.<br />
<br />
*Dr. '''Loren Looger''', Howard Hughes Medical Institute: for his use of computational protein design program to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. <br />
<br />
*Dr. '''Masayori Inouye''', Rutgers University: for the generous provision of Tar-EnvZ.<br />
<br />
*Dr. '''Luciano Marraffini''', Sontheimer Lab at Northwestern University, for the provision of plasmid pLM6, a shuttle vector for S.epidermidis/E.coli<br />
<br />
*Dr. '''Reinholb Bruckner''', for the provision of shuttle vectors PRB474 and PRB473<br />
<br />
<br />
----<br />
<br />
Thanks to '''Team Heidelberg''' for the inspiration for our Wiki menu design!<br />
<br />
----</div>Ashleyhttp://2009.igem.org/Team:Brown/TeamTeam:Brown/Team2009-10-21T16:47:36Z<p>Ashley: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
The Brown iGEM Lab is entirely student-run and consists of nine Brown University undergraduates. We began our training last spring with a lab course in Synthetic Biology. Under the gracious support of the Brown Undergraduate Teaching and Research Award (UTRA) Program and funding from various other academic departments, we were able to work throughout the summer on our project. Though the project's generation and implementation was an entirely student-directed process, the team owes much of its academic and research support to the faculty and graduate student advisers that helped make everything possible. Here at Brown, we pride ourselves in upholding the ideals set out by the iGEM competition, namely that the project we have set out to create is fully our own (from creation to completion) and that each student involved in the program be afforded his/her full opportunity to both learn and contribute in the lab. Therefore, in the true spirit of Synthetic Biology, our team's project this year works hard to reflect the many different research backgrounds contributed by its nine individual members; elements of electrical engineering, electrochemistry, genetics, and microbiology are incorporated. <br />
<br />
<br />
{|border = "0"<br />
|-<br />
|rowspan="0"|<br />
<br />
'''Advisors:'''<br />
<br />
*''' Faculty Advisor ''': Dr. Gary Wessel, Professor of Biology, Bio Med Molecular, Cellular Biology Biochemistry <br />
*'''Graduate Student Advisor ''': Adrian Reich, Graduate Student, Bio-Med (Bio) <br />
*'''Graduate Student Advisor ''': Diana Donovan, Graduate Student, Bio-Med (Bio)<br />
<br />
'''Undergraduate Students:'''<br />
<br />
*'''William Allen''': Will Allenquot<br />
*'''Michael Chang''': MC Mastermix<br />
*'''Stephanie Cheung''': Stephylococcus <br />
*'''Ashley Kim''': Ashley Kimwipe<br />
*'''Flora War War Ko''': Nasal Flora<br />
*'''Elias Scheer''': E.coli Scheer<br />
*'''Minoo Ramanathan''': Minoo Prep<br />
*'''Ahmad Rana''': Ahmad Ran-a-gel<br />
*'''Indu Voruganti''': The INDUcer<br />
<br />
|<br />
<gallery><br />
Image:Garywessel.jpg|Dr. Gary Wessel<br />
Image:Adrianreich.jpg|Adrian Reich<br />
</gallery><br />
<gallery><br />
Image:Will.jpg|Will Allen<br />
Image:Michael.jpg|Michael Chang<br />
Image:Steph.jpg|Steph Cheung<br />
Image:Ashley.jpg|Ashley Kim<br />
Image:flora.jpg|Flora Ko<br />
Image:eli.jpg|Eli Scheer<br />
Image:ahmad.jpg|Ahmad Rana<br />
Image:minoo.jpg|Minoo Ramanthan<br />
Image:indu.jpg|Indu Voruganti</div>Ashleyhttp://2009.igem.org/Team:Brown/ProjectTeam:Brown/Project2009-10-21T16:20:26Z<p>Ashley: /* A Synthetic Approach to Treating Allergic Rhinitis: Engineering Staphyloccocus Epidermidis to Secrete High-Affinity Histamine Binding Protein in Response to Elevated Levels of Histamine due to an Allergic Attack */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
== A Synthetic Approach to Treating Allergic Rhinitis: Engineering Staphyloccocus Epidermidis to Secrete High-Affinity Histamine Binding Protein in Response to Elevated Levels of Histamine during an Allergic Attack ==<br />
<br />
=Project Abstract=<br />
<br />
Brown University’s 2009 iGEM Team presents an exciting new approach to treating nasal allergies through Allergene: a synthetically engineered, self-regulating drug factory in the nose. This revolutionary new product provides a much-needed alternative to current antihistamines by directly sequestering the histamine released in an allergic response. In order to do this, Allergene makes use of the unique histamine binding protein rEV131, native to the tick Rhipicephalus appendiculatus. By taking advantage of rEV131's high binding affinity for histamine, Allergene effectively eliminates both the symptoms and side effects associated with allergies and their treatments. <br />
<br />
<br />
Rather than presenting a system that passively sequesters histamine; however, Allergene goes one step further to providing patients with much-needed allergy relief. Activation of the product only occurs in the explicit event of an actual allergic response. This ingenious mechanism is made possible through the novel engineering of a histamine receptor in prokaryotes, a grand undertaking that had never before been accomplished. By re-designing pre-existing prokaryotic chemoreceptors to bind histamine rather than their wild-type ligands, Allergene acquired its most impressive feature yet: the "Histamine Sensor". Site-directed mutagenesis on the ligand binding pockets of two particular prokaryotic chemoreceptors, Ribose Binding Protein and Tar (normally responsive to Aspartate)was performed to create this novel sensor. Through histamine detection, Allergene’s unique histamine sequestering system is engineered to function only when histamine levels are markedly high. This efficiently timed system is therefore completely self-regulating. <br />
<br />
<br />
Allergene is introduced to its host patients by capitalizing on the endogenous existence of bacterium Staphylococcus epidermidis in human nasal flora. This naturally present organism is the perfect vehicle for delivery; its rapid production and secretion of Allergene’s genetic constructs effectively implement the system’s histamine sensing and sequestering capabilities in human hosts. Secretion is accomplished through the attachment of a signal peptide sequence specific to S. epidermidis, a clear and concise method by which to deliver the ready-to-use drug. In order to eliminate any safety concerns associated with the utilization of S. epidermidis, special care has additionally been taken to engineer the product’s accessory kill switch mechanism. Capitalizing on the cells' abilities to sense the growth of their own populations, a DNA gyrase poison responsible for triggering cell death (CCDB)is neatly placed under the quorum or population sensing promoter Agr, which normally functions in hazardous biofilm formation. Named the “Quorum Sensor”, this thoughtful, additional feature prompts Allergene’s swift response in the unfavorable event of over-cell-proliferation.</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_S.epidermidisTeam:Brown/Project S.epidermidis2009-10-21T04:44:49Z<p>Ashley: /* The Chassis: Staphyloccocus Epidermidis */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=The Chassis: Staphyloccocus Epidermidis =<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Staphylococcus epidermidis is an ideal vehicle for our genetic machine for several reasons: it is a native organism in the human nasal flora, it is generally non-pathogenic, and its sequenced genome is used with some frequency in lab work.[[Image:Staphylococcus epidermidis.jpg|right|caption]]<br />
<br />
<br />
<br />
<br />
Since our gene of interest, rEV131, can only bind histamine if it is outside the cell, we have to engineer its secretion from S. epidermidis. We decided to use a signal peptide motif normally found on ß-lactamase, the S. epidermidis gene for ampicillin resistance, which uses the native Sec pathway for its secretion.<br />
We had the DNA sequence of this peptide synthesized and ligated it N-terminally to GFP as a test construct. This reporter would allow us to visually ascertain<br />
whether the produced protein was secreted. In our final genetic construct, the GFP reporter is replaced by rEV131.<br />
<br />
=Quorum Sensor=<br />
<br />
<br />
Although Staphylococcus epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell<br />
density becomes too great. This change in phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum.<br />
We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDB, a death gene.<br />
<br />
<br />
Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers use electroporation to induce cells to take up their target DNA. Throughout the course of our research, we have tweaked the electroporation protocol many times but there are always a very small number of transformants.</div>Ashleyhttp://2009.igem.org/Team:Brown/Project_S.epidermidisTeam:Brown/Project S.epidermidis2009-10-21T04:31:39Z<p>Ashley: /* The Chassis: Staphyloccocus Epidermidis */</p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=The Chassis: Staphyloccocus Epidermidis =<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Staphylococcus epidermidis is an ideal vehicle for our genetic machine for several reasons: it is a native organism in the human nasal flora, it is generally non-pathogenic, and its sequenced genome is used with some frequency in lab work.[[Image:Staphylococcus epidermidis.jpg|right|caption]]<br />
<br />
<br />
Since our gene of interest, rEV131, can only bind histamine if it is outside the cell, we have to engineer its secretion from S. epidermidis. We decided to use a signal peptide motif normally found on ß-lactamase, the S. epidermidis gene for ampicillin resistance, which uses the native Sec pathway for its secretion.<br />
We had the DNA sequence of this peptide synthesized and ligated it N-terminally to GFP as a test construct. This reporter would allow us to visually ascertain<br />
whether the produced protein was secreted. In our final genetic construct, the GFP reporter is replaced by rEV131. <br />
<br />
<br />
<br />
<br />
=Quorum Sensor=<br />
<br />
<br />
Although Staphylococcus epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell<br />
density becomes too great. This change in phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum.<br />
We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDB, a death gene.<br />
<br />
<br />
Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers use electroporation to induce cells to take up their target DNA. Throughout the course of our research, we have tweaked the electroporation protocol many times but there are always a very small number of transformants.</div>Ashleyhttp://2009.igem.org/Team:Brown/ProjectTeam:Brown/Project2009-10-21T03:58:02Z<p>Ashley: </p>
<hr />
<div>{{Brown}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
== A Synthetic Approach to Treating Allergic Rhinitis: Engineering Staphyloccocus Epidermidis to Secrete High-Affinity Histamine Binding Protein in Response to Elevated Levels of Histamine due to an Allergic Attack ==<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Project Abstract=<br />
<br />
Brown University’s 2009 iGEM Team presents an exciting new approach to treating nasal allergies through Allergene: a synthetically engineered, self-regulating drug factory in the nose. This revolutionary new product provides a much-needed alternative to current antihistamines by directly sequestering the histamine released in an allergic response. In order to do this, Allergene makes use of the unique histamine binding protein rEV131, native to the tick Rhipicephalus appendiculatus. By taking advantage of rEV131's high binding affinity for histamine, Allergene effectively eliminates both the symptoms and side effects associated with allergies and their treatments. <br />
<br />
<br />
Rather than presenting a system that passively sequesters histamine; however, Allergene goes one step further to providing patients with much-needed allergy relief. Activation of the product only occurs in the explicit event of an actual allergic response. This ingenious mechanism is made possible through the novel engineering of a histamine receptor in prokaryotes, a grand undertaking that had never before been accomplished. By re-designing pre-existing prokaryotic chemoreceptors to bind histamine rather than their wild-type ligands, Allergene acquired its most impressive feature yet: the "Histamine Sensor". Site-directed mutagenesis on the ligand binding pockets of two particular prokaryotic chemoreceptors, Ribose Binding Protein and Tar (normally responsive to Aspartate)was performed to create this novel sensor. Through histamine detection, Allergene’s unique histamine sequestering system is engineered to function only when histamine levels are markedly high. This efficiently timed system is therefore completely self-regulating. <br />
<br />
<br />
Allergene is introduced to its host patients by capitalizing on the endogenous existence of bacterium Staphylococcus epidermidis in human nasal flora. This naturally present organism is the perfect vehicle for delivery; its rapid production and secretion of Allergene’s genetic constructs effectively implement the system’s histamine sensing and sequestering capabilities in human hosts. Secretion is accomplished through the attachment of a signal peptide sequence specific to S. epidermidis, a clear and concise method by which to deliver the ready-to-use drug. In order to eliminate any safety concerns associated with the utilization of S. epidermidis, special care has additionally been taken to engineer the product’s accessory kill switch mechanism. Capitalizing on the cells' abilities to sense the growth of their own populations, a DNA gyrase poison responsible for triggering cell death (CCDB)is neatly placed under the quorum or population sensing promoter Agr, which normally functions in hazardous biofilm formation. Named the “Quorum Sensor”, this thoughtful, additional feature prompts Allergene’s swift response in the unfavorable event of over-cell-proliferation.</div>Ashley