BioBrick Circuit Mechanism
BioBrick Circuit Design
R BioBrick
E BioBrick
BioBrick Circuit Assembly
BioBrick Circuit Plasmid
BioBrick Lab 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 R+E BioBrick plasmid
8. Transform plasmid into an expression strain expressing lacI
9. Induce GFP expression with IPTG
10. Measure GFP fluorescence
What if we didn’t have BioBricks?
What if we didn’t have BioBricks?
Aequorea victoria (Jellyfish)
Extract genomic DNA
Design primers and
PCR amplify gfp gene
What if we didn’t have BioBricks?
Purify DNA
Sequence DNA
Digest gfp and vector DNA
What if we didn’t have BioBricks?
Ligate DNA
Sequence DNA
Transform DNA
Test for ampicillin resistance
Successful Transformants (plated on LB + Amp plates)
http://www.umext.maine.edu/onlinepubs/htmpubs/images/growcurve.gif
Shaking Incubator
Overnight cultures (grown in LB + Amp media)
Pelleted cells after 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: relaxed circular, linear,
supercoiled (covalently closed-circular)
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)
BioBricks Vector
Calculation of R BioBrick Restriction
R BioBrick: Cut with S & P
= 2079 – 14 + 55 = 2120 bp
SpeI
PstI
XbaI
Calculation of E BioBrick Restriction
E BioBrick: Cut with X & P
= 6 + 876 + 16 = 898 bp
SpeI
PstI
XbaI