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

Using Synthetic Biology and pClone Red for Authentic Research on Promoter

Function: Genetics (analyzing mutant promoters)

 Todd T. Eckdahl and A. Malcolm Campbell

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)

What is the consensus sequence for the two elements of a bacterial promoter?

-10 element = T80A95T45A60A50T96

Optimal position is -10, but position varies from -18 to -9 from center of -10 element to TSS at +1

-35 element = T82T84G78A65C54A45

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

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

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.

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

GGA Methodwith pClone Red

by A. Malcolm Campbell and Todd Eckdahl

G CTTAA AATTC G

Eco RI

type II

palindrome

G CTTAA AATTC G

Eco RI

type II

palindrome

G CTTAA AATTC G

Eco RI

type II

G CTTAA

AATTC G

Eco RI

type II

Bsa I

GAGACC CTCTGG

type IIs

not a palindrome

Bsa I

1234nGAGACC nCTCTGG

----

type IIs

----

Bsa I

1234nGAGACC nCTCTGG

type IIs

----

Bsa I

GGTCTCnCCAGAGn1234

type IIs

----

Bsa I

GGTCTCnCCAGAGn1234

type IIs

Bsa I1234nGAGACC ----nCTCTGG

GGTCTCn----CCAGAGn1234

cuts left

cuts right

pClone Red = Part J119137

BsaI cutsright

BsaI cutsleft

CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC

Bsa I Bsa IGCTG

GCGG

pClone Red Magnified

CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC

GCTGGCGG

pClone Red Promoter Removed

CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCCGCTG

GCGG

pClone Red Promoter Recloned

GCTGGCGG

CGAC(promoter) (promoter)CGCC

CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC

pClone Red Promoter Removed Again

GCTGGCGGCGAC(promoter)

(promoter)CGCC

CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC

Experimental Promoter Cloned in pClone Red

plasmid backbone

origin antibiotic resistance

RBS RFP

BsaI + Ligase

RBSGFP

pClone Red in GGA

plasmid backbone

origin antibiotic resistance

RBS RFP

BsaI + Ligase

RBSGFP

pClone Red after GGA

pClone Red + No Promoter

pClone Red only

notice only green colonies

pClone Red

pClone Red + Functional Promoter

notice red colonies

X pClone Red

pClone Red + Non-functional Promoter

notice non-green colonies

Registry of Standard Parts

Student Promoter (dnaKP1)

Student Promoter (dnaKP1)