(Difference between revisions)

Project

The process of high-throughput cloning is bottlenecked at the retriction and ligation stages. A combination of high costs, requirements for restriction site specific enzymes and general inefficiency of the process makes cloning on a large combinatorial gene library unviable. Circular Polymerase Extension Cloning (CPEC) addresses this issue by eliminating the need for restriction and ligation enzymes and thereby streamlining and condensing the procedure into the duration of 5 minutes. An extremely simple theory, CPEC piggybacks PCR in splicing genes. Figure 1 below outlines how CPEC may be used to insert a gene into a vector.

The gene insert is modified to have ends that overlap with the ends of the linearlized vector and both have similar melting temperatures. The insert and vector are placed within a PCR machine in the absence of primers. Denaturation separates the double-stranded insert and vector and the overlapping ends anneal. Polymerase extension mechanism is then used to complete the plasmid.

We will apply CPEC in the construction of a multi-component plasmid containing biobricks. Previous Duke iGEM projects have yielded the genes in a metabolic pathway that synthesizes poly(3HB-co-4HB), a biodegradable plastic, in E. coli. We will transform those genes into biobricks, with sticky ends, and efficiently combine them in a vector using CPEC.

Notebook

6/3/09
Experiment: PCR with contents of tube labeled 7 as DNA template, Gel with 100 bp ladder to check PCR
Results: Incorrect ladder and template
Conclusion: Should use 1 kb ladder, Need to find correct template

6/4/09
E: Gel to check Bioplastics #1-5
R: All correct sized bands
C: Use one as template for PCR

E: PCR with Bioplastics #5 as template

5x HF buffer 5 ul
dNTPs 2 ul
forward primer 1.25 ul
reverse primer 1.25 ul
DNA template (plasmid) 0.5 ul
Phusion DNA polymerase 0.3 ul
H2O 14.7 ul
25 ul

98°C 30s 1x
98°C 15s
55°C 30s
72°C 1m 10s 35x
72°C 5m
4°C infinity 1x

Fragment # Name Size/Length
1 pASKphaA 4.2 kb
2 phaB 900 bp
3 Termcat2 1.7 kb
4 phaC 1.9 kb

6/5/09
E: Gel to check PCR
R: Correct band for fragments 2 and 4, No band for fragments 1 and 3
C: Cut correct bands out of gel, Try PCR with another template, different conditions

E: PCR with Bioplastics #1 as template, increased extension time to 1m 20s

6/6/09
R: Correct band for fragments 1 (weak) and 2, No band for fragments 3 and 4
C: Cut out correct bands, Do PCR again with same template for fragments 1 and 2, Try PCR with different template for fragments 3 and 4

E: PCR with Bioplastics #1 (fragments 1 and 2) and #3 (fragments 3 and 4) as templates


6/8/09
E: Gel to check PCR, Gel with leftover PCR of right sized fragments
R: Correct bands for all fragments
C: Cut out bands

6/9/09
Gel Extraction
1. Weigh cut gel piece(s). Add at least 1 ul binding buffer per mg of gel.
2. Place tube at 50-60°C for 10-15 min or until gel is completely dissolved. Vortex every few minutes.
3. Add solution to spin column. Centrifuge at max speed for 30s. Discard filtrate.
4. Add 300 ul binding buffer to column. Centrifuge for 30s. Discard filtrate.
5. Add 500 ul wash buffer. Centrifuge for 30s. Discard filtrate.
6. Repeat step 5.
7. Centrifuge tubes for at least 2 min. Discard collection tube.
8. Obtain 1.7 ml centrifuge tube. Add 40-50 ul elution buffer to spin column. Centrifuge for 1 min. Discard spin column.

E: Measured concentration of DNA fragments
R: Very low concentrations of fragments 1 and 3
C: Do PCR with fragments as template, greater volume to increase amount

E: PCR with fragments 1 and 3 as templates

6/10/09
E: Gel to check PCR
R: Smears at bottom of gel
C: Should use Bioplastics #1 as template to increase amount of fragment 1 and Bioplastics #3 as template to increase amount of fragment

E: PCR with Bioplastics #1 and #3 as templates and doubled volume, Gel to obtain more of fragments
R: Smears for fragment 1, Correct bands for fragment 2, Correct bands and smears for fragment 3
C: PCR with different conditions

E: PCR with Bioplastics #3 as template, extension time increased to 1m 30s, cycles increased to 40

6/11/09
E: Gels to check PCR
R: Correct bands for fragment 1, 2 and 4 and possibly correct band for fragment 3
C: Cut out bands

E: Measured concentration of DNA fragments
R: Still low concentrations of fragments 1 and 3
C: Do more PCRs to obtain more of fragments 1 and 3

6/15/09
E: PCRs with Bioplastics #1 (fragments 1 and 2) and #3 (fragments 3 and 4) as templates, doubled volume, eliminated annealing step, varied extension (plus annealing) time based on fragment size, Gels to check PCRs

5x HF buffer 10 ul
dNTPs 4 ul
forward primer 2.5 ul
reverse primer 2.5 ul
DNA template (plasmid) 1 ul
Phusion DNA polymerase 0.8 ul
H2O 29.2 ul
50 ul

98°C 30s 1x
98°C 15s
72°C varies 35x
72°C 5m
4°C infinity 1x

Annealing Time: 30s
Extension Time: 15 s * size/length (kb)

Fragment # Name Size/Length (bp) Time (s)
1 pASKphaA 4215 100
2 phaB 896 50
3 Termcat2 1701 60
4 phaC 1872 65

R: Bands for fragments 1 and 2, No bands for fragments 3 and 4
C: Cut out bands of fragments 1 and 2, PCR again to obtain fragments 3 and 4

6/16/09
E: PCR with Bioplastics #2 as template and Phusion mix (done by Maggie), Gels to check PCRs

2x Phusion mix 25 ul
forward primer 2.5 ul
reverse primer 2.5 ul
DNA template (plasmid) 1 ul
H2O 19 ul
50 ul

R: No bands for fragments 3, Bands for fragment 4
C: Cut out bands of fragment 4, PCR again to obtain fragment 3

E: Measured concentration of DNA fragments 1 and 2
R: Concentrations okay
C: Do assembly PCR once enough of fragments 3 and 4 obtained

6/17/09
E: PCR with Bioplastics #4 as template, using Phusion enzyme protocol (shorter denaturation time, lower annealing temperature), Gel to check PCRs

98°C 15s 1x
98°C 1s
50°C 5s
72°C 30s 35x
72°C 2m
4°C infinity 1x

R: Correct bands
C: Cut out bands

E: Measured concentration of DNA fragments 3 and 4
R: Concentrations high
C: Do assembly PCR

6/18/09
E: Assembly PCR, Gels to check PCRs

2x Phusion mix 12.5 ul
insert 7.14 ul
H2O 5.36 ul
25 ul

98°C 30s 1x
98°C 10s
70-55°C 5s
72°C 30s 25x
72°C 2m
4°C infinity 1x

Fragment # Name Size/Length (bp) Concentration (ng/ul) Mass (ng) Volume (ul)
1 pASKphaA 4215 41 194 4.73
2 phaB 896 68 41 0.60
3 Termcat2 1701 69 78 1.13
4 phaC 1872 126 86 0.68
~400 7.14

Length of plasmid: 8360 bp + 4 Histags * 6 aa * 3 bp = 8432 bp

R: Smear because of misformed gel, no band
C: Change conditions

6/19/09
E: Assembly PCR (done by Maggie) with different conditions (decreased extension time, increased number of cycles)

5x HF buffer 5 ul
dNTPs 2 ul
insert 7.18 ul
Phusion DNA polymerase 0.4 ul
H2O 10.42 ul
25 ul

98°C 30s 1x
98°C 10s
70-55°C 30s
72°C 2m 40x
72°C 5m
4°C infinity 1x

Fragment # Name Mass (ng) Volume (ul)
1 pASKphaA 195.3 4.76
2 phaB 41 0.60
3 Termcat2 78.4 1.14
4 phaC 86.4 0.68
~400 7.14
6/20/09
E: Gel to check PCR
R: Correct band (faint)
C: Low efficiency, Use different method (Infusion kit or PCR with 2 fragments first)

6/22/09
LB Agar Plates with Chlorophenicol (CAM), anhydrotetracycline, Nile Red (NR)
stock plate
CAM 50 mg/ul 20 ug/ml
anhyd 2 mg/ml 150 ng/ml
NR 0.25 mg/ml 0.5 ug/ml

E: PCR with fragments 1 and 2 and different conditions (decreased annealing and extension times and number of cycles), Gel to check PCR

2x Phusion mix 12.5 ul
pASKphaA 4.76 ul
phaB 0.60 ul
H2O 7.14 ul
25 ul

98°C 30s 1x
98°C 10s
70-55°C 10s
72°C 1m 10s 20x
72°C 5m
4°C infinity 1x

R: No band/smear
C: Change conditions

6/23/09
E: Assembly PCR with different conditions (no slow ramp)

6/24/09
E: Gel to check PCR
R: Correct band (faint)
C: Do transformation with competent cells

6/24/09
Transformation with High Efficiency GC5 Competent Cells
1. Remove competent cells from -70°C and place on ice. Thaw for 5-10 min.
2. Gently mix cells by tapping tube.
3. Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl pipette tip while dispensing DNA. Gently tap tube to mix.
4. Place tubes on ice for 30 min.
5. Heat-shock cells for 45 sec in 42°C (water) bath. Do not shake!
6. Add 450 ul RT SOC Medium to each transformation reaction.
7. Incubate at 37°C for 1 hr with shaking at 225-250 rpm.
8. Spread on LB agar plates containing appropriate antibiotic.
9. Incubate plates at 37°C overnight (12-16 hrs).

6/26/09
Pick a colony from a streaked selective plate to inoculate 10 ml of LB medium supplemented with the appropriate selection antibiotic. Incubate 12-18 hrs at 37°C while shaking at 200-250 rpm.

6/27/09
Harvest bacterial culture by centrifuging at 8000 rpm (6800xg) in microcentrifuge for 2 min at RT. Decant supernatant and remove remaining medium.

Purification/Mini prep
1. Resuspend pelleted cells in 250 ul Resuspension Solution. Vortex or pipet up and down until no cell clumps remain.
2. Add 250 Lysis Solution and mix thoroughly by inverting tube 4-6 times until solution becomes viscous and slightly clear. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 ul Neutralization Solution and mix thoroughly by inverting tube 4-6 times.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer supernatant to spin column by decanting or pipetting. Avoid disturbing or transferring white precipitate.
6. Centrifuge for 1 min. Discard flow-through and replace column in collection tube.
7. Add 500 ul Wash Solution to spin column. Centrifuge for 30-60s and discard flow-through.
8. Repeat step 7.
9. Centrifuge for 1 min to remove residual Wash Solution.
10. Transfer spin column into fresh 1.7 ml microcentrifuge tube. Add 50 ul Elution Buffer to center of spin column membrane. Do not touch pipette tip to membrane. Incubate 2 min at RT and centrifuge for 2 min.
Note: An additional elution step with Elution buffer or water will recover residual DNA from the membrane and increase overall yield by 10-20%.
11. Discard column and store purified plasmid DNA at -20°C.

6/29/09
E: Restriction digests, Gel to check digests

Digest 1
NdeI (FastDigest) 1 ul
SpeI (NEB) 1 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Sizes/Lengths: 5 kb, 3 kb, 460 bp, (80bp)

Digest 2 (Did not do)
NdeI (FastDigest) 2 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Sizes/Lengths: 5.4 kb, 3 kb

Digest 3
BamHI (FastDigest) 1 ul
XhoI (FastDigest) 1 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Sizes/Lengths: 6.6 kb, 1.8 kb

Digest 4
BamHI (FastDigest) 2 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Size/Length: 8.4 kb

R: Correct bands and sizes for plasmid 2 and digests 1, 3, and 4, correct band and size for plasmid 4
C: Do more tests on plasmid 2 such as sequencing to determine if plasmid is carryover, Try different primers

About our Team

Advisors

Dr. Jingdong Tian

Undergraduates

{| |[[Image:Sahil.jpg|200px]] |'''Sahil "Mr Baller" Prasada'''
A remarkable individual of considerable talent, ability, and girth, the esteemed Mr. Prasada is the latest addition to Duke's elite iGEM team. His innumerable achievments span many varied fields, earning numerous recognitions at innovation summits (for his humor), at tennis tourneys (for his egregious sportsmanship), and at local dances (for his exaggerated gyrations of the hip) among others. His research interests are likewise broad, and as one of the best and brightest young minds of our time, he is very excited to share his latest findings with you at iGEM this November. Sahil aspires to be a cardiologist, just like his father, when he grows up. His favorite hobbies include tennis, kangaroo-watching, eating, and bhangra dancing. Sahil's academic interests include yeast mating, doxorubicin, and hydrogen fuels. |- |[[Image:kchien.jpg|200px]] |'''Kevin Chien'''
A reformed Carolina fan. |}

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