1. 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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

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

22. 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

23. pClone Red + Non-functional PromoterX
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

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

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

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