WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to...
Transcript of WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to...
![Page 1: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/1.jpg)
![Page 2: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/2.jpg)
WHAT’S T-REX?
• Control the synthesis of any protein of interest
• Silence the protein expression faster than using classic regulated promoters
iGEM2009–UniversityofBologna
![Page 3: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/3.jpg)
This device is composed of two BioBricks:
• TRANS- repressor
• CIS- repressing
iGEM2009–UniversityofBologna
![Page 4: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/4.jpg)
• Transcription of the target gene yields a mRNA strand;
• The mRNA with the CIS sequence at 5' end, is available for
translation.
iGEM2009–UniversityofBologna
![Page 5: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/5.jpg)
• When the promoter controlling the TRANS coding sequence is active its transcript binds with the CIS mRNA.
• This RNA duplex prevents ribosomes from binding to RBS, thus silencing protein synthesis.
iGEM2009–UniversityofBologna
![Page 6: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/6.jpg)
TESTING CIRCUIT
iGEM2009–UniversityofBologna
O2
![Page 7: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/7.jpg)
iGEM2009–UniversityofBologna
1) Maximalfreeenergyinthesecondarystructure,reducingtheprobabilityofitsintra‐molecularannealing;
2) Minimalunwantedinterac?onswithgenomicmRNA;
3) Minimalprobabilityofpar?al/shiEedhybridiza?onwithcomplementarystrands.
BASER Best Sequence Research by Andrea and Elisa
![Page 8: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/8.jpg)
iGEM2009–UniversityofBologna
HOW BASER WORKS?
Star?ngfromarandomlygeneratedsequence(currentsequence);
Conformitytest:a) morethan5adjacentrepeatsofthesamenucleo?de;b) restric?onsites;c) RBSsequences;
NO
YES
![Page 9: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/9.jpg)
iGEM2009–UniversityofBologna
BASERreplace5nucleto?desrandomly(genera?onofnewsequence);
Evaluatescoreofnewsequence;
AddRBSat3’end
Evaluatescoreofcurrentsequence;
ScoreofnewsequenceisbeSerthanscoreofcurrent
sequence?
Newsequenceispreserved
Currentsequenceispreserved
NO YES
![Page 10: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/10.jpg)
iGEM2009–UniversityofBologna
• BASER calculates a score for the current sequence:
1) the self score;
2) the genomic score;
3) the shifted score;
HowBASERcalculatesthescore?
![Page 11: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/11.jpg)
iGEM2009–UniversityofBologna
ChooseofaCISsequence
AACACAAACTATCACTTTAACAACACATTACATATACATTAAAATATTACAAAGAGGAGAAA(RBSinitalic)
![Page 12: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/12.jpg)
iGEM2009–UniversityofBologna
CCTCTTTGTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTTwitha7b‐longRBScoveringreen
ChooseoftheTRANSsequences
CTTTGTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTTwitha4b‐longRBScoveringreenunderlined
![Page 13: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/13.jpg)
iGEM2009–UniversityofBologna
Morphology:‐Eccentricity[0,1];‐Area[min,max];
Focus:‐Clustering;‐Highfluorescence;‐Highcellnumber;
Output:foreachbacteriumtheareainpixelsandthefluorescence
VIFluoR
![Page 14: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/14.jpg)
iGEM2009–UniversityofBologna
Part Characterization
• PromoterStrengths
• Plasmidcopynumbers
• InfluenceofO2operator
• Interac?onbetweenLacIandO2operator
pSB3K3 pSB1A2 BBa_J23100
BBa_K07919 BBa_B0034
BBa_J0431 BBa_B0015 BBa_J23100 BBa_B0015
BBa_J23118 BBa_C0012 BBa_B0015
![Page 15: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/15.jpg)
iGEM2009–UniversityofBologna
Promoter Strengths
BBa_J23118 (1429)
BBa_K079032 on pSB1A2
BBa_J23100 BBa_B0034 BBa_J04031 BBa_B0015
BBa_K079031 on pSB1A2
BBa_J23118 BBa_B0034 BBa_J04031 BBa_B0015
vsBBa_J23100
(2547)
![Page 16: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/16.jpg)
iGEM2009–UniversityofBologna
Promoter Strengths • Methods
‐DH5αcells‐M9medium‐37°overnight
• ImagingAnalysis‐VIFluoR
• FluorimeterAnalysis‐TecanM200
BBa_J23118
BBa_J23100
![Page 17: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/17.jpg)
iGEM2009–UniversityofBologna
Promoter Strengths • OD/Fluorescenceover?meanalysisfromOD=0.1au
‐ GrowthCurve‐ Fluorescence‐ Fluorescence/ODra?o
![Page 18: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/18.jpg)
iGEM2009–UniversityofBologna
Plasmid Copy Numbers
pSB1A2 (high copy) vs pSB3K3
(low/medium copy)
BBa_K201003 on pSB1A2
BBa_B0034 BBa_E0040 BBa_B0015 BBa_J23118
BBa_K201003 on pSB3K3
BBa_B0034 BBa_E0040 BBa_B0015 BBa_J23118
![Page 19: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/19.jpg)
• Methods‐DH5αcells‐M9medium‐37°overnight
pSB3K3
pSB1A2
Plasmid Copy Numbers
iGEM2009–UniversityofBologna
• ImagingAnalysis‐VIFluoR
• FluorimeterAnalysis‐TecanM200
![Page 20: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/20.jpg)
iGEM2009–UniversityofBologna
Influence of O2
BBa_K079032 (O2 absent) vsBBa_K201001
(O2 present)
BBa_K079032 on pSB1A2
BBa_B0034 BBa_E0040 BBa_B0015 BBa_J23100
BBa_K201001 on pSB1A2
BBa_B0034 BBa_E0040 BBa_B0015 BBa_J23100 BBa_K07919
![Page 21: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/21.jpg)
• Methods‐DH5αcell‐M9medium‐37°overnight
iGEM2009–UniversityofBologna
• FluorimeterAnalysis‐Victor2
pSB1A2
Influence of O2
![Page 22: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/22.jpg)
iGEM2009–UniversityofBologna
Positive Control of Testing Circuit
BBa_K201002 on pSB3K3 BBa_B0034 BBa_C0012 BBa_B0015 BBa_J23118 BBa_B0034 BBa_B0015 BBa_J23100
BBa_K201001 on pSB1A2 BBa_k07919
![Page 23: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/23.jpg)
iGEM2009–UniversityofBologna
• ImagingAnalysis‐ VIFluoR‐ severalimages‐ >60bacteria/image
IPTG induction: Static Response • Methods
‐DH5αcells‐M9medium‐ 37°overnight‐ severalIPTGlevels
![Page 24: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/24.jpg)
iGEM2009–UniversityofBologna
• FluorimeterAnalysis‐TecanM200‐ Dilu?ontoOD=0.1‐ 1°sample:NoIPTG‐ 2°sample:IPTG100μM
• GrowthCurve• Fluorescence
• Methods‐DH5αcell‐M9medium‐ 37°overnight‐ NoIPTG
IPTG induction: Dynamic Response
![Page 25: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/25.jpg)
iGEM2009–UniversityofBologna
MATHEMATICAL MODEL • Transcrip?onandtransla?onprocesseswereconsideredsimilartoasecondorderkine?cs,likeanenzyma?creac?on:
![Page 26: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/26.jpg)
iGEM2009–UniversityofBologna
MATHEMATICAL MODEL
![Page 27: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/27.jpg)
iGEM2009–UniversityofBologna
![Page 28: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/28.jpg)
iGEM2009–UniversityofBologna
![Page 29: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/29.jpg)
iGEM2009–UniversityofBologna
PARAMETERS ASSIGNMENT FromLiterature
![Page 30: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/30.jpg)
FromExperimentalMeasurement
iGEM2009–UniversityofBologna
PARAMETERS ASSIGNMENT
PROMOTERRATIO=1.2
PLASMIDCOPYNUMBERRATIO=4.6
Wesimulatedtes?ngcircuitwhenT‐REXdeviceisidle(Ini?alTrans‐DNA=0)
![Page 31: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/31.jpg)
iGEM2009–UniversityofBologna
LacI SIGMOIDAL REPRESSION CURVE
![Page 32: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/32.jpg)
iGEM2009–UniversityofBologna
WefiSedexperimentaldatainordertoiden?fyLacI‐O2dissocia?onconstantandLacI‐IPTGdissocia?onconstant
STATIC IPTG INDUCTION
![Page 33: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/33.jpg)
iGEM2009–UniversityofBologna
DYNAMIC IPTG INDUCTION
Fiqngofthe100µMIPTGdynamicinduc?onwith?me‐varyingRNApolymerase
![Page 34: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/34.jpg)
iGEM2009–UniversityofBologna
T-REX SIMULATION
![Page 35: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/35.jpg)
iGEM2009–UniversityofBologna
T-REX STORY Wedidn’tmanagetogetthefinalcircuitbecausewedidn’tachieve
theassemblyingoftheCISandTRANSparts
Whichweretheproblems?
1. Partsareonly100bpinlength:Quan?typroblem,duetopurifica?on?
*P1010deathgeneliga?onprotocol.
2. Enzymeefficencyislowerwithshortflankingsequences:Wereourdiges?onseffec?ve?
*WeorderlongerPCRprimersanddoubledthediges?on?me.
![Page 36: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/36.jpg)
CONCLUSIONS
Enterinforma?ondetailingatleastonenewstandardBioBrickPartorDeviceintheRegistryofStandardPartsanddemonstratethatworksasexpected;
SubmitDNAforatleastonenewBioBrickPartorDevicetotheRegistryofParts.
iGEM2009–UniversityofBologna
![Page 37: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/37.jpg)
CONCLUSIONS
Characterizeorimproveanexis?ngBioBrickPartorDeviceandenterthisinforma?onbackontheRegistry.
HelpanotheriGEMteam:ClonedandsenttheBioBrickBBa_K201002totheUNIPV‐PaviaiGEMteam.
iGEM2009–UniversityofBologna
![Page 38: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/38.jpg)
HUMAN PRACTICE- SHARING
We realized:
• An Online Survey
• An Information Booklet
iGEM2009–UniversityofBologna
![Page 39: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/39.jpg)
ON-LINE SURVEY RESULTS
• General lack of knowledge about Synthetic Biology
• Most people expressed curiosity about Synthetic Biology and iGEM
• After reading the booklet, great part of the respondent recognized the importance of a responsible and conscious use of Synthetic Biology
iGEM2009–UniversityofBologna
Totalrespondents:484
![Page 40: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/40.jpg)
Acknowledgements
• University of Bologna
• Ser.In.Ar Cesena
• Cultural Association San Sebastiano
Instructors: Silvio Cavalcanti, Francesca Ceroni, Emanuele Domenico Giordano, Alejandro
Hochkoeppler, Marco Caprini
iGEM2009–UniversityofBologna
![Page 41: WHAT’S T-REX?2009.igem.org/files/presentation/Bologna.pdf · T-REX STORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and](https://reader033.fdocuments.in/reader033/viewer/2022042119/5e98f1a5611cac583d2db77b/html5/thumbnails/41.jpg)
iGEM2009–UniversityofBologna