CLONING AND EXPRESSION OF NEUTRAL PROTEASE GENE FROM B. STEAROTHERMOPHILUS

1.Introduction of initial project 2.Generally overview 3.Flow of Experiment 4.Results and Conclusions TABLE OF CONTENTS

 GOI: nprT  nprT neutral thermostable protease  Size of gene: 1881 bp  Designing the cloning model for the production of thermostable neutral protease  Bio brick Part chosen: BBa_K09112 INTRODUCTION OF INITIAL PROJECT

Extraction of Bacterial genome PCR Primers F- Primer R- Electrophoresis T-Vector ligation Transformation in E.coli cells Sequencing PCR with Biobrick compatible primers nprT Bio brick Construction Transformation into E.coli cells Detection of Enzyme Promoter selection and transformation Isolation of Plasmid Restriction digestion Glycerol stock OVERVIEW OF PROJECT

Trial 1Trial 2 PROMOTERS CONSTRUCTION

Trial 3 We have used 3 bio brick parts 1.BBa_K091112(2009 Amp r ): pLacIQ1 promoter 2.BBa_I0500(2011 Kan r ): Inducible pBad/araC promoter 3.BBa_K206001(2011 Amp r ): pBAD weak PROMOTERS CONSTRUCTION

Trial 1  Restriction enzymes: X+P combination  Expected size: 130 bp Trial 2  Restriction enzymes: I.X+S combination II.E+P combination  Expected Size:130 bp RESTRICTION DIGESTION X+s E+p +ve 100bp bp 100bp

Trial tubes of culture. 2.Not enough growth observed in overnight 3.Unable to visualize DNA precipitation Trial 2 1.Inoculated two new tubes 2.3 tubes were two week old 3.Electrophoresis. EXTRACTION OF BS CHROMOSOMAL DNA

ELECTROPHORESIS OF EXTRACTED DNA 43 B3 21 Ladder

Primer usedBPTm( o C)ΔGΔGGC% For 5’ ATG AAC AAA CGG GCG ATG C 1957+ve ( )52.6 Rev 5’ TTA ATA CAC TCC AAC CGC ATT G 2254-ve (0.33)40.9 Biobrick-For 5’ GAATTCGCGGCCGCTTCTAG ATG AAC AAA CGG GCG ATG C ve ( )56.4 Biobrick-Rev 5’ TACTAGTAGCGGCCGCTGCAG TTA ATA CAC TCC AAC CGC ATT G ve ( )51.2 PCR 5’ATGAACAAACGGGCGATGC 3’ 5’ATGAACAAACGGGCGATGCTCGGGGCGATCGGGCTGGCGTTCTTCGGCGAAGGGGGAAT CGATCGTCTGGAACG…………………………………………TACTATTTGACGCCGACGTCGA ACTTCGTGCCGCCTGCGTGCAAGCGGCCGCTGATTTGTACGGGTCGACAAGCCAAGAAGT CAACTCGGTGAAACAGGCGTTCAATGCGGTTGGAGTGTATTAA 3’ 3’ GTTACGCCAACC TCACATAATT 5’

Trial 1 PCR Why continue to Trial 2? Ladder 2(45.0) 2 (48.6) 2(52.9)2(55.0) B3 (45.0) B3 (48.6) B3 (52.9) B3 (55.0) +ve -ve 1900bp

Trial 2 Ladder 1(38.0)1(38.7)1(40.0)1(42.0)1(44.6)1(46.7) 1(48.1) 1(49.0) B3 +ve -ve 1900bp

Trial 3 No nprT Continue with exp. +ve -ve B3(59.1) B3(57.6) B3(55.3)B3(52.4)B3(50.3)B3(48.8) Ladder

Two ways of purification: Intensity of bands  Direct PCR Product Purification: labeled A to D  Gel Purification: labeled 1 to 13 PCR PRODUCTS PURIFICATION PCR Trial 1 PCR Trial 2PCR Trial 3 1AB C D

GEL PURIFICATION bp

PURIFICATION CONFIRMATION 1500bp 200bp Ladder Direct

Trial 1Trial 2 LIGATION AND TRANSFORMATION

 Total of 8 colonies from selected plates only  Subjected for overnight growth  Plasmid extraction and send for sequencing EXTRACTION & SEQUENCING Transformation- 2 PlatesColonies WhiteBlue Many

Alignment Matches:  2 sequences showing alignment with Cloning vector pGBT-R16  Both Blue colonies SEQUENCING DATA

Alignment Matches:  6 sequences showing alignment with Anoxybacillus flavithermus WK1, complete genome SEQUENCING DATA

 Unsuccessful cloning of nprT gene.  Successful cloning of Bio brick promoter BBa_K  Successful cloning of unknown gene. Anoxybacillus flavithermus WK1 CONCLUSION