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

WHAT’S T-REX?

•  Control the synthesis of any protein of interest

•  Silence the protein expression faster than using classic regulated promoters

iGEM2009–UniversityofBologna

This device is composed of two BioBricks:

•  TRANS- repressor

•  CIS- repressing


•  Transcription of the target gene yields a mRNA strand;

•  The mRNA with the CIS sequence at 5' end, is available for

translation.


•  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.


TESTING CIRCUIT


O2


1)  Maximalfreeenergyinthesecondarystructure,reducingtheprobabilityofitsintra‐molecularannealing;

2)  Minimalunwantedinterac?onswithgenomicmRNA;

3)  Minimalprobabilityofpar?al/shiEedhybridiza?onwithcomplementarystrands.

BASER Best Sequence Research by Andrea and Elisa


HOW BASER WORKS?

Star?ngfromarandomlygeneratedsequence(currentsequence);

Conformitytest:a) morethan5adjacentrepeatsofthesamenucleo?de;b) restric?onsites;c) RBSsequences;

NO

YES


BASERreplace5nucleto?desrandomly(genera?onofnewsequence);

Evaluatescoreofnewsequence;

AddRBSat3’end

Evaluatescoreofcurrentsequence;

ScoreofnewsequenceisbeSerthanscoreofcurrent

sequence?

Newsequenceispreserved

Currentsequenceispreserved

NO YES


•  BASER calculates a score for the current sequence:

1) the self score;

2) the genomic score;

3) the shifted score;

HowBASERcalculatesthescore?


ChooseofaCISsequence

AACACAAACTATCACTTTAACAACACATTACATATACATTAAAATATTACAAAGAGGAGAAA(RBSinitalic)


CCTCTTTGTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTTwitha7b‐longRBScoveringreen

ChooseoftheTRANSsequences

CTTTGTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTTwitha4b‐longRBScoveringreenunderlined


Morphology:‐Eccentricity[0,1];‐Area[min,max];

Focus:‐Clustering;‐Highfluorescence;‐Highcellnumber;

Output:foreachbacteriumtheareainpixelsandthefluorescence

VIFluoR


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


Promoter Strengths

BBa_J23118 (1429)

BBa_K079032 on pSB1A2




vsBBa_J23100

(2547)


Promoter Strengths •  Methods

‐DH5αcells‐M9medium‐37°overnight

•  ImagingAnalysis‐VIFluoR

•  FluorimeterAnalysis‐TecanM200

BBa_J23118

BBa_J23100


Promoter Strengths •  OD/Fluorescenceover?meanalysisfromOD=0.1au

‐ GrowthCurve‐ Fluorescence‐ Fluorescence/ODra?o


Plasmid Copy Numbers

pSB1A2 (high copy) vs pSB3K3

(low/medium copy)


BBa_B0034 BBa_E0040 BBa_B0015 BBa_J23118

BBa_K201003 on pSB3K3


•  Methods‐DH5αcells‐M9medium‐37°overnight

pSB3K3

pSB1A2

Plasmid Copy Numbers


•  ImagingAnalysis‐VIFluoR

•  FluorimeterAnalysis‐TecanM200


Influence of O2

BBa_K079032 (O2 absent) vsBBa_K201001

(O2 present)




BBa_B0034 BBa_E0040 BBa_B0015 BBa_J23100 BBa_K07919

•  Methods‐DH5αcell‐M9medium‐37°overnight


•  FluorimeterAnalysis‐Victor2

pSB1A2

Influence of O2


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


•  ImagingAnalysis‐ VIFluoR‐ severalimages‐ >60bacteria/image

IPTG induction: Static Response •  Methods

‐DH5αcells‐M9medium‐ 37°overnight‐ severalIPTGlevels


•  FluorimeterAnalysis‐TecanM200‐ Dilu?ontoOD=0.1‐ 1°sample:NoIPTG‐ 2°sample:IPTG100μM

• GrowthCurve• Fluorescence

•  Methods‐DH5αcell‐M9medium‐ 37°overnight‐ NoIPTG

IPTG induction: Dynamic Response


MATHEMATICAL MODEL • Transcrip?onandtransla?onprocesseswereconsideredsimilartoasecondorderkine?cs,likeanenzyma?creac?on:


MATHEMATICAL MODEL




PARAMETERS ASSIGNMENT FromLiterature

FromExperimentalMeasurement


PARAMETERS ASSIGNMENT

PROMOTERRATIO=1.2

PLASMIDCOPYNUMBERRATIO=4.6

Wesimulatedtes?ngcircuitwhenT‐REXdeviceisidle(Ini?alTrans‐DNA=0)


LacI SIGMOIDAL REPRESSION CURVE


WefiSedexperimentaldatainordertoiden?fyLacI‐O2dissocia?onconstantandLacI‐IPTGdissocia?onconstant

STATIC IPTG INDUCTION


DYNAMIC IPTG INDUCTION

Fiqngofthe100µMIPTGdynamicinduc?onwith?me‐varyingRNApolymerase


T-REX SIMULATION


T-REX STORY Wedidn’tmanagetogetthefinalcircuitbecausewedidn’tachieve

theassemblyingoftheCISandTRANSparts

Whichweretheproblems?

1. Partsareonly100bpinlength:Quan?typroblem,duetopurifica?on?

*P1010deathgeneliga?onprotocol.

2. Enzymeefficencyislowerwithshortflankingsequences:Wereourdiges?onseffec?ve?

*WeorderlongerPCRprimersanddoubledthediges?on?me.

CONCLUSIONS

Enterinforma?ondetailingatleastonenewstandardBioBrickPartorDeviceintheRegistryofStandardPartsanddemonstratethatworksasexpected;

SubmitDNAforatleastonenewBioBrickPartorDevicetotheRegistryofParts.


CONCLUSIONS

Characterizeorimproveanexis?ngBioBrickPartorDeviceandenterthisinforma?onbackontheRegistry.

HelpanotheriGEMteam:ClonedandsenttheBioBrickBBa_K201002totheUNIPV‐PaviaiGEMteam.


HUMAN PRACTICE- SHARING

We realized:

•  An Online Survey

•  An Information Booklet


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


Totalrespondents:484

Acknowledgements

•  University of Bologna

•  Ser.In.Ar Cesena

•  Cultural Association San Sebastiano

Instructors: Silvio Cavalcanti, Francesca Ceroni, Emanuele Domenico Giordano, Alejandro

Hochkoeppler, Marco Caprini



Documents

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