1. CLONING AND EXPRESSION OF NEUTRAL PROTEASE GENE FROM B. STEAROTHERMOPHILUS

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

3.  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

4. 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

5. Trial 1Trial 2 PROMOTERS CONSTRUCTION

6. 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

7. 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 9 10 5 7243 8 1 1 172 3 4 5 89 3000bp 100bp

8. Trial 1 1.8 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

9. ELECTROPHORESIS OF EXTRACTED DNA 43 B3 21 Ladder

10. Primer usedBPTm( o C)ΔGΔGGC% For 5’ ATG AAC AAA CGG GCG ATG C 1957+ve (0.64- 1.34)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 3969.5-ve (3.08-6.04)56.4 Biobrick-Rev 5’ TACTAGTAGCGGCCGCTGCAG TTA ATA CAC TCC AAC CGC ATT G 4368.4-ve (1.75-3.39)51.2 PCR 5’ATGAACAAACGGGCGATGC 3’ 5’ATGAACAAACGGGCGATGCTCGGGGCGATCGGGCTGGCGTTCTTCGGCGAAGGGGGAAT CGATCGTCTGGAACG…………………………………………TACTATTTGACGCCGACGTCGA ACTTCGTGCCGCCTGCGTGCAAGCGGCCGCTGATTTGTACGGGTCGACAAGCCAAGAAGT CAACTCGGTGAAACAGGCGTTCAATGCGGTTGGAGTGTATTAA 3’ 3’ GTTACGCCAACC TCACATAATT 5’

11. 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

12. 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

13. 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

14. 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 2 9 8 76543 C D 10 11 12 13

15. GEL PURIFICATION 2 1 3 4 7 6 5 13 12 11 10 1 3 2 4 5 678 9 100bp

16. PURIFICATION CONFIRMATION 1500bp 200bp Ladder 1234567 Direct

17. Trial 1Trial 2 LIGATION AND TRANSFORMATION

18.  Total of 8 colonies from selected plates only  Subjected for overnight growth  Plasmid extraction and send for sequencing EXTRACTION & SEQUENCING Transformation- 2 PlatesColonies WhiteBlue 102 200 300 400 506 602 700 8158 938 10013 11Many0 1200 1300

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

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

21.  Unsuccessful cloning of nprT gene.  Successful cloning of Bio brick promoter BBa_K206001  Successful cloning of unknown gene. Anoxybacillus flavithermus WK1 CONCLUSION