A. Malcolm Campbell and Todd T. Eckdahl Using Synthetic Biology and pClone Red for Authentic Research on Promoter Function: Introductory Biology (identifying new promoters)   A. Malcolm Campbell and Todd T. Eckdahl

A. Malcolm Campbell and Todd Eckdahl GGA Method with pClone Red This slide show gives students more details about how GGA works. by A. Malcolm Campbell and Todd Eckdahl

Eco RI AATTC G G CTTAA palindrome type II EcoRI is a type II restriction enzyme. Type II recognition sequences are palindromes. AATTC G G CTTAA palindrome type II

Eco RI AATTC G G CTTAA palindrome type II EcoRI is a type II restriction enzyme. Type II recognition sequences are palindromes. AATTC G G CTTAA palindrome type II

Eco RI AATTC G G CTTAA type II EcoRI cuts along the purple line to produce a palindromic, 4-base sticky end. AATTC G G CTTAA type II

Eco RI G CTTAA AATTC G type II EcoRI cuts along the purple line to produce a palindromic, 4-base sticky end. G CTTAA AATTC G type II

Bsa I GAGACC CTCTGG not a palindrome type IIs BsaI is a type IIS restriction enzyme. Type IIs recognition sequences are NOT palindromes. GAGACC CTCTGG not a palindrome type IIs

Bsa I ---- 1234nGAGACC nCTCTGG type IIs Type IIs enzymes cut to one side or the other of the recognition sequence. Here BsaI cuts to the left of the recognition sequence as shown. ---- 1234nGAGACC nCTCTGG type IIs

Bsa I ---- 1234nGAGACC nCTCTGG type IIs Type IIS sticky ends can vary in length and they often skip one or more bases outside their recognition sequence as shown here by a pair of n bases. The sticky ends (shown as 1234) will vary depending on what sequences are adjacent to the recognition sequence. The variable sticky ends is what makes them so helpful in GGA. ---- 1234nGAGACC nCTCTGG type IIs

Bsa I GGTCTCn CCAGAGn1234 ---- type IIs Here the recognition sequence is shown as the reverse compliment of the previous slide. Now the enzyme cuts to the right and produces a different sticky end. GGTCTCn CCAGAGn1234 ---- type IIs

Bsa I GGTCTCn CCAGAGn1234 ---- type IIs BsaI always produces a 5’ overhang. GGTCTCn CCAGAGn1234 ---- type IIs

Bsa I 1234nGAGACC ----nCTCTGG GGTCTCn---- CCAGAGn1234 cuts left cuts You can compare the two versions of the BsaI recognition site, cuts left or cuts right. The only difference is the directionality of the recognition site. cuts left GGTCTCn---- CCAGAGn1234 cuts right

pClone Red = Part J119137 BsaI BsaI cuts cuts left right pClone Red contains two opposing BsaI sites such that both recognition sites will be removed when digested at both sites. BsaI cuts left BsaI cuts right

pClone Red Magnified Bsa I Bsa I CGACtGAGACC GGTCTCa GCGG Zoomed in view of pClone Red shows the base pairs flanking the default, left-facing promoter (PlacIQ1). CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC GCGG GCTG Bsa I Bsa I

pClone Red Promoter Removed CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC When BsaI cuts on both sides of the promoter, you can see the sticky ends produced when the promoter is removed. Notice that the BsaI recognition sites are removed at the same time. GCGG GCTG

pClone Red Promoter Recloned The original promoter can ligate back in because ligase is present throughout GGA. But if the promoter ligates in, it can be cut out again. CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC GCGG GCTG

pClone Red Promoter Removed Again CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC Again, the original promoter is removed and this time the students’ experimental promoter will replace it. Notice both have the same sticky ends and the cloning will be directional. Notice that the experimental promoter cannot contain a BsaI recognition site. GCGG GCTG CGAC(promoter) (promoter)CGCC

Experimental Promoter Cloned in pClone Red CGACtGAGACC GGTCTCa aCTCTGG CCAGAGtCGCC This time, the experimental promoter is ligated and cannot be removed by BsaI. CGAC(promoter) (promoter)CGCC GCGG GCTG

antibiotic resistance pClone Red in GGA RFP GFP RBS RBS This slide shows the overall starting place of pClone Red (J119137) with the original promoter in place. BsaI + Ligase antibiotic resistance origin plasmid backbone

antibiotic resistance pClone Red after GGA RFP GFP RBS RBS After GGA, the original promoter is removed and the experimental promoter has taken its place. BsaI + Ligase antibiotic resistance origin plasmid backbone

pClone Red + No Promoter only When pClone Red is transformed into cells, all the colonies are green. notice only green colonies

pClone Red + Functional Promoter If a functional promoter is cloned, a subset of the colonies will be red. In this example, about half of the colonies are red. notice red colonies

pClone Red + Non-functional Promoter X pClone Red If a non-functional promoter replaces the original promoter, then about half of the colonies will be colorless (not green). The green colonies still contain the original promoter. notice non-green colonies

Registry of Standard Parts Examples of student promoters submitted to the Registry of Standard Parts at the iGEM web site.

Student Promoter (dnaKP1) One specific example is show. This one does not have sequences annotated, which is not ideal.

Student Promoter (dnaKP1) Experimental data showing how well the experimental promoter works compared to the + control plasmid J04450.