randomized biobrick assembly: a novel dna assembly method ... · sean c. sleight* and herbert m....
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Randomized BioBrick Assembly:A novel DNA assembly method for randomizing and optimizing
multi-gene circuits and metabolic pathways
Sean C. Sleight* and Herbert M. SauroUniversity of Washington, Dept. of Bioengineering, Seattle, WA 98195, USA
Synthetic biology requires DNA synthesis or the assembly of genetic parts into functional genetic circuits and metabolic pathways. The optimization of circuits and pathways often requires constructing various iterations of the same construct, or directed evolution to achieve the desired function. Alternatively, a method that randomizes individual parts in the same assembly reaction could be used for optimization by allowing for the ability to screen large numbers of individual clones expressing randomized circuits or pathways for optimal function. Here we describe a new assembly method to randomize genetic circuits and metabolic pathways from modular DNA fragments derived from PCR-amplified BioBricks. Each fragment of a particular part type (e.g. promoters, coding sequences, and transcriptional terminators) has the same standardized overlap on either side of the functional DNA, allowing for independent assembly with other fragments having the complementary overlap. When multiple fragments of a particular type are used in the same assembly reaction, there is competition between fragments, allowing for randomized assembly. As a proof-of-principle for this method, we first assembled eCFP, maxRFP, and eYFP gene expression cassettes with independently randomized promoters, ribosome binding sites, transcriptional terminators, and all parts randomized at once. These randomized expression cassettes were then combined to make fully functional and randomized three-gene circuits that express CFP, RFP, and YFP, producing colors close to Cyan, Magenta, and Yellow (CMY) under UV light. Sequencing results from 12 CMY circuits with nine randomized terminators show that 9/12 circuits are distinct and at least one circuit contains each of the nine terminators. When all parts are shuffled at once, 11/12 circuits are distinct and expression ranges from about 2 to 160-fold above background levels depending on the fluorescent protein. We then adapted this method to randomize the same promoters, ribosome binding sites, and terminators with the enzyme coding sequences (crtE, crtB, and crtI) from the lycopene biosynthesis pathway instead of fluorescent proteins, designed to allow each enzyme in the pathway to be independentally controlled from a different promoter. Sequencing results show that 7/8 pathways with all parts shuffled at once are distinct and lycopene production can be controlled with different inducer concentrations. Although the sample size is low, the randomized lycopene pathways have a strikingly different frequency of promoters and RBSs relative to the CMY circuit. These results demonstrate the ability to generate multiple unique three-gene circuits and pathways in the same assembly reaction, allowing for the construction of large libraries that can be subjected to high-throughput selections and screens. We expect that this randomization method will be useful for increasing DNA assembly efficiency, optimizing metabolic flux to maximize products of interest (e.g. biofuels), and is likely adaptable to other circuits and pathways.
METHODOLOGY
SEQUENCING RESULTS
OPTIMIZATION AND CHARACTERIZATION
ACKNOWLEDGEMENTSWe thank BEACON: An NSF Center for the Study of Evolution in Action for funding this research, the Registry of Standard Biological Parts and DNA2.0 for parts, SBOL Visual V1.0 for circuit diagrams, the UW Department of Bioengineering, and Sauro Lab (Wilbert Copeland, Bryan Bartley, Michal Galdzicki, Kyung Kim, Greg Medlock especially) and Klavins Lab (Rob Egbert and Josh Bishop) members for useful materials and discussions. Special thanks to Rob Egbert for the maxRFP construct and to the 2011 UW iGEM team for the pGA3K3 vector!
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0.400
0.500
0.600
0.700
R0010 R0040 R0062
Freq
uenc
y of
Par
t / T
otal
Pro
mot
er P
arts
CMYLycopene
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
Freq
uenc
y of
Par
t / T
otal
Ter
min
ator
Par
ts
CMYLycopene
Frequency of Promoters in an All-Part 3-Cassette Assembly Frequency of RBSs in an All-Part 3-Cassette Assembly Frequency of Terminators in an All-Part 3-Cassette Assembly
R0040
eYFP
B0024
J61048
bla
maxRFP B0015
rrnB1
B0014
ScarBScarAA B
eCFP B0011
B0025rpn
ScarCC D
J61048
J61048
J61048
J61048
J61048
J61048
J61048
J61048
B0024
1
2
3
4
5
6
7
8
9
10
11
12
Frequency of Promoters, RBSs, and Terminators in 12 plasmids that express eYFP, maxRFP, and eCFP
R0010
R0010
R0062
R0010
R0062
R0010
R0062
R0040
R0062
R0010
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
R0010
R0062
eYFP
J61048
J61048
J61048
B0015
B0015
B0015*
R0062
R0062
R0062
R0062
R0062
R0062
R0010
R0062
R0010
R0062
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
R0062
R0010
maxRFP
B0014
B0015
B0012
B0014
B0015
B0015-11
B0015
B0015
B0015
rpn
rpn
R0062
R0062
R0010
R0062
R0062
R0062
R0010
R0062
R0062
R0062
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
R0010
R0062
eCFP
rpn
rpn
rpn
B0025
B0025
B0011
B0011
B0011
B0025
rpn
R0010
R0062
B0035B0034
J15001B0035B0034
J15001
B0035B0034
J15001
B0035
B0035
B0035
J15001
B0035
J15001
B0034
B0034
B0035
J15001
B0035
B0035
B0035
B0035
B0034
B0035
B0034
B0035
B0034-35
B0034
B0035
B0034
B0034
B0035
B0034
B0034
B0034
B0034
B0034
B0035
B0034
J15001
B0034
J15001
B0035-34
B0034-35
R0040R0010
R0062
R0040R0010
R0062
R0040 eYFP
B0024
J61048
bla
R0010 maxRFP B0015
rrnB1
B0014
B0035 ScarBB0034 ScarAA B
R0062 eCFP B0011
B0025rpn
J15001 ScarCC D
bla
J61048
B0024
J61048
J61048
J61048
bla
J61048
bla
1
2
3
4
5
6
7
8
9
10
11
12
Frequency of Terminators in 12 plasmids that express eYFP, maxRFP, and eCFP
R0040
R0040
R0040
R0040
R0040
R0040
R0040
R0040
R0040
R0040
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
eYFP
R0040
R0040
eYFP
bla
J61048
bla
rrnB1
B0014
B0015
R0010
R0010
R0010
R0010
R0010
R0010
R0010
R0010
R0010
R0010
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
maxRFP
R0010
R0010
maxRFP
rrnB1
rrnB1
B0015
B0015
B0015
B0015
B0015-rrnB1***
B0015
rrnB1
rpn
B0011
R0062
R0062
R0062
R0062
R0062
R0062
R0062
R0062
R0062
R0062
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
eCFP
R0062
R0062
eCFP
B0025
rpn
rpn
rpn
rpn
rpn
B0011
B0011
B0011
rpn
R0040 pTetR eYFP
B0024
J61048
bla
pGA3K3-araC-luxR-VectorAB
R0010 pLacI maxRFP B0015
rrnB1
B0014
pGA3K3-araC-luxR-VectorBC
R0062 pLuxR eCFP B0011
B0025
rpn
pGA3K3-araC-luxR-VectorCD
J15001 ScarC
B0035 ScarB
B0034 ScarAA B
B C
C D
R0040 pTetR eYFP
B0024
J61048
bla
R0010 pLacI maxRFP
B0015
rrnB1
B0014
B0035 ScarB
B0034 ScarAA
B
R0062 pLuxR eCFP B0011
B0025rpn
J15001 ScarC
C
D
B
C
pGA3K3-araC-luxR-VectorAD
A D
eYFP circuit with randomized terminators A, B, and C
maxRFP circuit with randomized terminators D, E, and F
eCFP circuit with randomized terminators G, H, and I
Assembly results
Pool of randomized circuits PCR-amplified with A and B primers
No-insert vector PCR-amplified with A and D primersA 4-way assembly reaction with the this vector and 3 insertsabove are transformed into E. coli TURBO cells (to the left).
Step 2: assemble plasmids with randomized parts (e.g. terminators)
Steps 3 and 4: PCR and 4-way assembly reaction
+
+
+
=3-gene circuit with randomized terminators A-I
Pool of randomized circuits PCR-amplified with B and C primers
Pool of randomized circuits PCR-amplified with C and D primers
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12
Indu
ced
/ U
nind
uced
YFP
Flu
ores
cenc
e
All Parts Randomized CMY Circuit #
0
20
40
60
80
100
120
140
1 2 3 4 5 6 7 8 9 10 11 12
Indu
ced
/ U
nind
uced
RFP
Flu
ores
cenc
e
All Parts Randomized CMY Circuit #
0
2
4
6
8
10
12
14
1 2 3 4 5 6 7 8 9 10 11 12
Indu
ced
/ U
nind
uced
RFP
Flu
ores
cenc
e
All Parts Randomized CMY Circuit #
YFP, RFP, and CFP expression above background in CMY circuits with all parts randomized
R0010 pLacI
R0040 pTetR
R0062 pLuxR
eYFP
eCFP
maxRFP
J61048
B0024
bla
pGA3K3-araC-luxR
21bp 21bp18bp 18bp 20bp 20bp 20bp 20bp
PCRPCRPCRPCR
RBS RBS
RBS RBS
RBS RBS
Step 1: PCR-amplify parts with standaridzed overlaps
Inducer experiment to measure expression in CMY circuits and Lycopene pathways with all parts randomized+ AHL + AHL + IPTG CMY Circuit # + IPTG Lycopene Pathway #+ aTc 2 3 4 5 6 7 9 10 + aTc 1 2 3 4 5 6 7 C1
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
0
1 2 3 4 5 6 7 8 Std. C
12 parts randomized at once:4 promoters, 4 fluorescent protein coding sequences,4 transcriptional terminators
1
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
0
Lycopene cell pellets
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0.050
0.100
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0.200
0.250
0.300
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0.400
0.450
B0034 B0035 J15001 B0034-35 B0035-34
Freq
uenc
y of
Par
t / T
otal
RBS
Par
ts
CMYLycopene
R0040
crtE
B0024
J61048
bla
crtB B0015
rrnB1
B0014
ScarBScarAA B
B0011
B0025rpn
ScarCC D
B0024
J61048
J61048
bla
B0024
B0024
1
2
3
4
5
6
7
8
Frequency of Promoters, RBSs, and Terminators in 12 plasmids that express lycopene
R0010
R0040*
R0062
R0040
R0062
R0062
R0062
R0062
crtE
crtE
crtE
crtE
crtE
crtE
crtE
crtE
B0015
B0015
B0014
R0040*
R0010
R0010
R0040*
R0010
R0010
R0010
R0010
crtB
crtB
crtB
crtB
crtB
crtB
crtB
crtB
B0015
rrnB1
rrnB1
B0012
rrnB1
rpn
B0025
R0062
R0010
R0062-40**
R0010
R0010
R0010
R0040*
R0010
crtI
crtI
crtI
crtI
crtI
crtI
crtI
crtI
B0025
B0025
B0025
B0025
B0025
rpn
R0010
R0062
B0035B0034
J15001B0035B0034
J15001
B0035B0034
J15001
B0034
B0034
B0034
J15001
J15001
B0035
J15001
J15001
B0035
J15001
B0034
B0035
B0034-35
B0035
B0034
J15001
J15001
J15001
J15001
B0035
R0040R0010
R0062
R0040R0010
R0062
crtI
B0034-35 J61048
bla
B0035-34
B0035
J15001