WHAT’S T-REX? • Control the synthesis of any protein of interest • Silence the protein expression faster than using classic regulated promoters iGEM 2009 – University of Bologna
This device is composed of two BioBricks: • CIS- repressing • TRANS- repressor iGEM 2009 – University of Bologna
• Transcription of the target gene yields a mRNA strand; • The mRNA with the CIS sequence at 5' end, is available for translation. iGEM 2009 – University of Bologna
• 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. iGEM 2009 – University of Bologna
TESTING CIRCUIT O2 iGEM 2009 – University of Bologna
BASER Best Sequence Research by Andrea and Elisa 1) Maximal free energy in the secondary structure, reducing the probability of its intra‐molecular annealing; 2) Minimal unwanted interac?ons with genomic mRNA; 3) Minimal probability of par?al/shiEed hybridiza?on with complementary strands. iGEM 2009 – University of Bologna
HOW BASER WORKS? Star?ng from a randomly generated sequence (current sequence); Conformity test: a) more than 5 adjacent NO repeats of the same nucleo?de; b) restric?on sites; c) RBS sequences; YES iGEM 2009 – University of Bologna
Add RBS at 3’ end Evaluate score of current sequence; BASER replace 5 nucleto?des randomly (genera?on of new sequence); Evaluate score of new sequence; Score of new Current YES sequence is NO New sequence beSer than sequence is is preserved score of preserved current sequence? iGEM 2009 – University of Bologna
How BASER calculates the score? • BASER calculates a score for the current sequence: 1) the self score; 2) the genomic score; 3) the shifted score; iGEM 2009 – University of Bologna
Choose of a CIS sequence AACACAAACTATCACTTTAACAACACATTACATATACATTAAAATATTAC AAAGAGGAGAAA (RBS in italic ) iGEM 2009 – University of Bologna
Choose of the TRANS sequences CCTCTTT GTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTT with a 7b‐long RBS cover in green CTTT GTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTT with a 4b‐long RBS cover in green underlined iGEM 2009 – University of Bologna
VIFluoR Morphology: ‐ Eccentricity [0,1]; ‐ Area [min,max]; Focus: ‐ Clustering; ‐ High fluorescence; ‐ High cell number; Output: for each bacterium the area in pixels and the fluorescence iGEM 2009 – University of Bologna
Par art Char t Characteriza acterization tion BBa_J23118 BBa_C0012 BBa_B0015 BBa_J23100 BBa_B0034 BBa_B0015 BBa_J23100 BBa_B0015 pSB3K3 BBa_K07919 BBa_J0431 pSB1A2 • Promoter Strengths • Plasmid copy numbers • Influence of O2 operator • Interac?on between LacI and O2 operator iGEM 2009 – University of Bologna
Promoter Str Pr omoter Strengths engths BBa_J23100 BBa_J23100 BBa_J23118 BBa_J23118 vs (2547) (2547) (1429) (1429) BBa_B0034 BBa_B0015 BBa_J23100 BBa_B0034 BBa_B0015 BBa_J23118 BBa_J04031 BBa_J04031 BBa_K079031 on pSB1A2 BBa_K079032 on pSB1A2 iGEM 2009 – University of Bologna
Promoter Str Pr omoter Strengths engths Methods • ‐ DH5α cells ‐ M9 medium ‐ 37° overnight BBa_J23118 Imaging Analysis • ‐ VIFluoR • Fluorimeter Analysis BBa_J23100 ‐ Tecan M200 iGEM 2009 – University of Bologna
Pr Promoter Str omoter Strengths engths OD/Fluorescence over?me analysis from OD=0.1au • ‐ Growth Curve ‐ Fluorescence ‐ Fluorescence/OD ra?o iGEM 2009 – University of Bologna
Plasmid Cop Plasmid Copy Number y Numbers s pSB3K3 pSB3K3 pSB1A2 pSB1A2 vs (lo (low/medium cop w/medium copy) y) (high cop (high copy) y) BBa_J23118 BBa_B0034 BBa_B0015 BBa_J23118 BBa_B0034 BBa_B0015 BBa_E0040 BBa_E0040 BBa_K201003 on pSB1A2 BBa_K201003 on pSB3K3 iGEM 2009 – University of Bologna
Plasmid Copy Number Plasmid Cop y Numbers s Methods • ‐ DH5α cells ‐ M9 medium ‐ 37° overnight pSB1A2 Imaging Analysis • ‐ VIFluoR • Fluorimeter Analysis ‐ Tecan M200 pSB3K3 iGEM 2009 – University of Bologna
Influence of O2 Influence of O2 vs BBa_K201001 BBa_K201001 BBa_K079032 BBa_K079032 (O2 present) (O2 pr esent) (O2 absent) (O2 a bsent) BBa_J23100 BBa_B0034 BBa_B0034 BBa_B0015 BBa_B0015 BBa_J23100 BBa_K07919 BBa_E0040 BBa_E0040 BBa_K079032 on pSB1A2 BBa_K201001 on pSB1A2 iGEM 2009 – University of Bologna
Influence of Influence of O2 O2 Methods • ‐ DH5α cell ‐ M9 medium ‐ 37° overnight pSB1A2 • Fluorimeter Analysis ‐ Victor 2 iGEM 2009 – University of Bologna
Positiv ositive Contr e Control of ol of T Testing Cir esting Circuit cuit BBa_J23100 BBa_J23118 BBa_C0012 BBa_B0015 BBa_B0034 BBa_B0015 BBa_B0034 BBa_k07919 BBa_K201002 on pSB3K3 BBa_K201001 on pSB1A2 iGEM 2009 – University of Bologna
IPTG induction: Sta IPT G induction: Static R tic Response esponse Methods • ‐ DH5α cells ‐ M9 medium ‐ 37° overnight ‐ several IPTG levels Imaging Analysis • ‐ VIFluoR ‐ several images ‐ >60 bacteria/image iGEM 2009 – University of Bologna
IPT IPTG induction: Dynamic R G induction: Dynamic Response esponse Fluorimeter Analysis • Methods • ‐ Tecan M200 ‐ DH5α cell ‐ Dilu?on to OD=0.1 ‐ M9 medium ‐ 1° sample: No IPTG ‐ 37° overnight ‐ 2° sample: IPTG 100μM ‐ No IPTG • Growth Curve • Fluorescence iGEM 2009 – University of Bologna
MATHEMATICAL MODEL • Transcrip?on and transla?on processes were considered similar to a second order kine?cs, like an enzyma?c reac?on: iGEM 2009 – University of Bologna
MATHEMATICAL MODEL iGEM 2009 – University of Bologna
iGEM 2009 – University of Bologna
iGEM 2009 – University of Bologna
PARAMETERS ASSIGNMENT From Literature iGEM 2009 – University of Bologna
PARAMETERS ASSIGNMENT From Experimental Measurement PROMOTER RATIO =1.2 PLASMID COPY NUMBER RATIO=4.6 We simulated tes?ng circuit when T‐REX device is idle (Ini?al Trans‐DNA = 0) iGEM 2009 – University of Bologna
LacI SIGMOIDAL REPRESSION CURVE iGEM 2009 – University of Bologna
STATIC IPTG INDUCTION We fiSed experimental data in order to iden?fy LacI‐O2 dissocia?on constant and LacI‐IPTG dissocia?on constant iGEM 2009 – University of Bologna
DYNAMIC IPTG INDUCTION Fiqng of the 100 µM IPTG dynamic induc?on with ?me‐varying RNA polymerase iGEM 2009 – University of Bologna
T-REX SIMULATION iGEM 2009 – University of Bologna
T-REX ST -REX STOR ORY We didn’t manage to get the final circuit because we didn’t achieve the assemblying of the CIS and TRANS parts Which were the problems? 1. Parts are only 100 bp in length: Quan?ty problem, due to purifica?on? * P1010 death gene liga?on protocol. 2. Enzyme efficency is lower with short flanking sequences: Were our diges?ons effec?ve? * We order longer PCR primers and doubled the diges?on ?me. iGEM 2009 – University of Bologna
CONCLUSIONS Enter informa?on detailing at least one new standard BioBrick Part or Device in the Registry of Standard Parts and demonstrate that works as expected; Submit DNA for at least one new BioBrick Part or Device to the Registry of Parts. iGEM 2009 – University of Bologna
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