BioBrick Assembly Methods
Standard assembly
In-Fusion PCR assembly
Using standard assembly to construct
a simple GFP circuit
BioBrick Circuit Mechanism
BioBrick Circuit Plasmid
Standard Assembly Overview
1. Amplify R and E BioBrick plasmids by transformation into a
cloning strain
2. Pick a single colony for overnight growth
3. Isolate plasmids using a miniprep kit
4. Run plasmids on a gel
5. Restriction digest of plasmids
6. Run digests on a gel and isolate the fragments of interest
7. Ligate fragments together to form the engineered plasmid
8. Transform plasmid into an expression strain expressing lacI
9. Induce GFP expression with IPTG
10. Measure GFP fluorescence with plate reader or
fluorescence microscope
Transformation via electroporation
Successful Transformants (plated on LB + Amp plates)
http://www.umext.maine.edu/onlinepubs/htmpubs/images/growcurve.gif
Overnight cultures (grown in LB + Amp media)
First step of a miniprep:
pellet cells with centrifugation
Rules of Gel Electrophoresis
• DNA runs towards the positive pole
• Smaller DNA fragments travel faster
than larger ones
• Electrophoretic mobility from slowest
to fastest: nicked open-circular,
relaxed circular, linear,
supercoiled (covalently closed-circular)‫‏‬,
supercoiled denatured
E-gel
Purified BioBrick plasmids
1 = Purified R plasmid (Sean)
2 = Purified E plasmid (Sean)
3 = Purified R plasmid (Alec)
4 = Purified E plasmid (Alec)
M = E-gel ladder
5 = 1 kb ladder
BioBrick Circuit Assembly
BioBricks Plasmid Digest
1 = 1 kb ladder
2 = Digested R plasmid (Sean)
3 = Undigested R plasmid
4 = Digested R plasmid (Alec)
M = E-gel ladder (10 uL)
5 = E-gel ladder (25 uL)
6 = Digested E plasmid (Sean)
7 = Undigested E plasmid
8 = Digested E plasmid (Alec)
Next we need to ligate the R-vector with the E-insert
with DNA Ligase
Ligation of R and E BioBricks
ACTAGT
TGATCA
XbaI
Mixed
SpeI
A
TGATC
ACTAGA
TGATCT
CTAGA
T
PstI
PstI
CTGCAG
GACGTC
TCTAGA
AGATCT
TGCAG
C
CTGCAG
GACGTC
C
GACGT
CTGCAG
GACGTC
Measuring DNA concentration for ligation reaction
We want 10 ng of vector mass. How much insert mass do we need?
Calculation of R BioBrick Restriction
R BioBrick: Cut with S & P
= 2079 – 14 + 55 = 2120 bp
SpeI
PstI
EcoRI
XbaI
Calculation of E BioBrick Restriction
E BioBrick: Cut with X & P
= 6 + 876 + 16 = 898 bp
SpeI
PstI
EcoRI
XbaI
Measuring DNA concentration for ligation reaction
We want 10 ng of vector mass. How much insert mass do we need?
R-vector = 7.5 ng/ul
E-insert = 18.4 ng/ul
25.4 ng E-insert = 6 X (898 / 2120) X 10 ng R-vector
10 ng R-vector / 7.5 ng/ul = 1.33 ul
25.4 ng E-insert / 18.4 ng/ul = 1.38 ul
Expression strain
That expresses lacI
Successful Transformants
Also, it is essential to miniprep the plasmid and sequence it.
GFP-expressing colonies
Fluorescence microscopy photo of
R0011+E0240 circuit in E. coli induced with IPTG