1. Using Synthetic Biology and pClone Red for Authentic Research on Promoter Function: Genetics (analyzing mutant promoters) Todd T. Eckdahl and A. Malcolm Campbell

2. Timeline Learn about bacterial promoters Develop promoter mutation hypotheses Order oligonucleotides for mutant promoters Use Golden Gate assembly to clone mutant promoters upstream of RFP reporter gene Measure output of reporter gene Present results to class and enter into Registry of Functional Reporters (RFP)

3. What is the consensus sequence for the two elements of a bacterial promoter? -10 element = T 80 A 95 T 45 A 60 A 50 T 96 Optimal position is -10, but position varies from -18 to -9 from center of -10 element to TSS at +1 -35 element = T 82 T 84 G 78 A 65 C 54 A 45 Position varies, but spacing between -35 and -10 elements is 16-18 bp in 90% of known promoters Optimal spacing is 17 bp Source: Genes IX, 2008, by Benjamin Lewin

4. Transcription Initiation RNAP binds to DNA and engages in direct exchange of one sequence for another Affinity of RNAP for nonspecific DNA decreased by Sigma factor RNAP + Sigma “touches down” at -35 element Contact is extended to -10 element, covering ~77 bp in “closed” binary complex Melting is facillitated by A+T content of -10 element - of ~12 bp from -10 element to +1 produces “open” binary complex Incorporation of NTPs forms ternary complex, and multiple rounds of abortive initiation occur Change in RNAP structure occurs, Sigma factor is released or changes form, and it covers ~50 bp RNAP clears the promoter, shortens to cover only 30-40 bp, and elongation occurs at a rate of ~40 nt/sec

5. pTac Promoter Hybrid promoter constructed from pTrp and pLac promoters in E. coli Efficiency of promotion measure to be 2-3 times great than pTrp and 7-11 times greater than pLac Perfect matches to -1- and -35 consensus sequences and 16 bp between them -35 -10 +1 DeBoer HA, Comstock LJ, Vasser M (1982) PNAS 80, 21-25.

6. Mutations of pTac Substitutions -10 element -35 element Deletions – Decrease spacing between -35 and -10 – Decrease spacing beteen -10 and +1 Insertions – Increase spacing between -35 and -10 – Increase spacing beteen -10 and +1

7. GGA Method with pClone Red by A. Malcolm Campbell and Todd Eckdahl

8. G CTTAA AATTC G Eco RI type II palindrome

9. G CTTAA AATTC G Eco RI type II palindrome

10. G CTTAA AATTC G Eco RI type II

11. G CTTAA AATTC G Eco RI type II

12. Bsa I GAGAC C CTCTG G type IIs not a palindrome

13. Bsa I 1234nGAGAC C nCTCTGG ---- type IIs

14. ---- Bsa I 1234nGAGAC C nCTCTGG type IIs

15. ---- Bsa I GGTCTCn CCAGAGn1234 type IIs

16. ---- Bsa I GGTCTCn CCAGAGn1234 type IIs

17. Bsa I 1234nGAGAC C ----nCTCTGG GGTCTCn---- CCAGAGn123 4 cuts left cuts right

18. pClone Red = Part J119137 BsaI cuts right BsaI cuts left

19. CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC Bsa I GCT G GCG G pClone Red Magnified

20. CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC GCT G GCG G pClone Red Promoter Removed

21. CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC GCT G GCG G pClone Red Promoter Recloned

22. GCT G GCG G CGAC(promoter) (promoter)CGCC CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC pClone Red Promoter Removed Again

23. GCT G GCGG CGAC(promoter) (promoter)CGCC CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC Experimental Promoter Cloned in pClone Red

24. plasmid backbone origin antibiotic resistance RBS RFP BsaI + Ligase RBS GFP pClone Red in GGA

25. plasmid backbone origin antibiotic resistance RBS RFP BsaI + Ligase RBS GFP pClone Red after GGA

26. pClone Red + No Promoter pClone Red only notice only green colonies

27. pClone Red pClone Red + Functional Promoter notice red colonies

28. X pClone Red pClone Red + Non-functional Promoter notice non-green colonies

29. Registry of Standard Parts

30. Student Promoter (dnaKP1)