Team:NTU Taida/LOGBOOK

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Latest revision as of 01:10, 18 October 2014

NTU-Taida

JUNE

6/24

mRNA extraction from Arabidopsis thaliana
Learn how to extract the plasmid.
Learn how to conduct the gel extraction and electrophoresis
Learn how to transform.
Learn how to do the gel extraction
Learn how to use the PCR Machine.
Design the primer for the FadL,R

6/25

RNA gel electrophoresis
Result: Failed (Smeared band indicated degradation of mRNA)

6/30

mRNA extraction for the second time
Result: Failed (Smeared band indicated degradation of mRNA)
Design the primer for the FadL,R
Determine whether to use the elongated primer(primer which contains the first 20 mer of FadR,L coding sequence and plus the restriction site which contains the E,X,S,P.
After heavily discussion, we decided to use the elongated primer. Though there is a big chance of failure, but it’s easy to operate.
Visit the 2014 Igem website and learn the rules set by the Igem.
Learn the biobrick design.

JULY

7/1

mRNA extraction for the third time
Result: Failed (Smeared band indicated degradation of mRNA)
Use the primer we designed to get the FadR, L from E.coli.
Failed 4 times. But the fifth cloning experiment had a huge success.
Therefore, we have the Fad R/L, though they had already registered in the iGEM website(sample not in stock)

7/2

mRNA extraction for the fourth time
Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana)
Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)
Primer design: CCD1、LOX1、HPL1
HPL1 has to be point mutated in order to become suitable for IGEM backbone pSB1C3

7/15

PCR(CCD1、LOX1、HPL1)
Result: Failed
Using the biobrick, and transformed the biobrick plasmid to our DH5-alpha E.coli.
1. Ribosomal binding site: RBS30.RB34
2. Promoter: J23100, J23101
3. Terminator: B0015, B0012
4. Plasmid backbone: Psb1A3, Psb1C3, Psb1K3
5. GFP: E0034, E0040
Extract the plasmid and make them into stocks.
Design the PKEK sequence, with whole genome synthesis (But later this idea was disapproved by the professor an use the primer synthesis method)
Make discussion with dry lab member to determine which strategy should use for modeling our pathway. Later we all agree to use the GFP assay.

7/17

PCR(CCD1、LOX1、HPL1)
Gradient: 50°C~60°C
Result: Failed (Probably there are some problem with our cDNA)
Started to assemble the gene circuit.
1. The beginning of every circuit is to cut the RBS 30/34 with restriction enzymes SpeI & PstI. And the FadR/L is cut with XbaI and PstI restriction enzymes.
2. After digestion for four hours, we conducted the electrophoresis and gel extraction for our target DNA sequences.
3. We ligated the RBS30/34 and FadR/L with ligase.(ligation high2.0)
Overnight.
4. Transformed the overnight plasmid to the DH5alpha competent cell.
5. Colony PCR to make sure whether we had insert the right gene into our competent cells.

7/18

Transformation of the following biobrick parts:
B0033 (RBS, weak), C0051 (CI), R0051 (pCI), B0015 (terminator)
All of them using CP plate.
Results: R0051 (pCI) failed.

7/19

Bacterial culture: B0033 (RBS, weak), C0051 (CI), B0015 (terminator)
Redo R0051 (pCI) transformation =>failed again
Possible reason: We mistakenly took the plasmid backbone of R0051 as pSB1C3, which is actually pSB1A2, and therefore used the wrong antibiotics. On the next day, we used Amp+ plate, add more SOB in recovery, use another competent cell(XBL-blue), and did it again.
mRNA extraction for the fifth time
Result: Failed (Smeared band indicated degradation of mRNA)
mRNA extraction for the sixth time
Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana)
Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)

7/20

Plasmid extraction: B0033 (RBS33, weak), C0051 (CI), B0015 (terminator)
(1、2 is terminator，3、4 is RBS， 5、6 is CI)
redo R0051 (pCI) transformation =>success!
PCR(CCD1、LOX1、HPL1)
Gradient: 50°C~60°C
Result: Succeeded with CCD1 at annealing temperature of 55°C, Failed with LOX1 and HPL1
Gel extraction and purification (CCD1)

7/21

Restriction enzyme digestion of RBS33, CI, and terminator, using PstI and EcoRI.
Then use DNA electrophoresis to confirm that all of them are correct.
bacterial culture of pCI.
PCR(LOX1、HPL1)
Result: Succeeded with one of HPL1’s point-mutated parts at annealing temperature of 52°C. Failed with LOX1 and HPL1’s another point-mutated parts.
Gel extraction and purification (one of HPL1’s point-mutated parts)

7/22

Plasmid extraction of pCI and confirmation, using EcoRI digestion and DNA electrophoresis. The DNA concentration was too low that band was invisible on the agarose gel.
PCR(LOX1、HPL1)
Gradient: 50°C~60°C
Result: Failed with both LOX1 and HPL1
Primer design: (FAD6、HPL1 redesign)

7/23

Redo bacterial culture and plasmid extraction of pCI. Failed again.
Possible reason: We didn’t wait for a few minutes for MP2 to react, so the cell lysis step wasn’t complete.
Another project is to make the GFP assay. So we decided to ligate the RBS 34/30 with GFP. The method are identical to the Fad R/L gene circuits.
For now we have:
1. RBS30 – FadR
2. RBS30 – FadL
3. RBS34 – FadR
4. RBS34 – FadL
5. RBS34 – GFP
6. RBS30 – GFP

7/24

PCR(LOX1、HPL1’s another part)
Gradient: 45°C~65°C
Result: Failed with both LOX1 and HPL1

7/28

Redo bacterial culture and plasmid extraction of pCI.
Redo transformation of pCI using another competent cell, DH5-alpha.
bacterial culture of Bacillus licheniformis at 30 ℃, using nutrient broth.
mRNA extraction for the seventh time
Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana)
Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)

7/29

plasmid extraction of pCI succeeded, but DNA electrophoresis failed, there was no band near 2.1k. Reason unknown.
Extract whole genome from Bacillus licheniformis. Failed.
Possible reason:
1. Bacillus licheniformis grows slower than E. coli. We didn’t give it enough time to grow.
2. Bacillus licheniformis is gram+, its thick cell wall may be harder to lyse.=> use another protocol.
PCR(LOX1、HPL1’s another part)
Gradient: 50°C~60°C
Result: Failed with both LOX1 and HPL1

7/30

Redo bacterial culture of Bacillus licheniformis =>The bacteria didn’t grow.
Subculture of Bacillus licheniformis plate. =>success
Redo pCI DNA electrophoresis. There was an unclear band near 2.1k.
PCR(FAD6)
Gradient: 50°C~60°C
Result: Succeeded with FAD6 at annealing temperature of 58°C
Gel extraction and purification (FAD6)

7/31

Redo bacterial culture of Bacillus licheniformis =>The medium dried out because the cap didn’t fit.
Another project is to make the GFP assay. So we decided to ligate the RBS 34/30 with GFP. The method are identical to the Fad R/L gene circuits.
For now we have:
1. RBS30 – FadR
2. RBS30 – FadL
3. RBS34 – FadR
4. RBS34 – FadL
5. RBS34 – GFP
6. RBS30 – GFP
And we started to ligate the promoter part to the existing gene circuits.
Prepare the presentation and PPT for the 2014 Asia Igem conference.
The laboratory was polluted by a strange strain of E.coli, and it seems like this polluted bacteria also contained the RFP, which appears in our every electrophoresis result. But this strange phenomenon suddenly seized in the beginning of August
We decided to make our PKEK gene circuits by primer-annealing methods. And we ordered the primers and materials in the late July.

AUGUST

Attending the Igem Asia conference, exchanged our thought of signal sequence and PKEK. We got lots of inspiration and suggestion, which influenced our latter experiment methods and design.
We decided to bring our PKEK circuit in to commercial design. By fusion our PKEK circuits to the his-tag, we can order our E.coli to produce huge amounts of PKEK, and use the his-tag to catch our PKEK product. (This part is still under conistruction)
In order to make our Igem PKEK gene circuit more practical, we combined commercial competition with our PKEK project. We wanted to use the synthetic biology to produce huge amounts of medical peptides, which have high value and can make contribution to people all over the world. Therefore, we participated the China Innovation & Entrepreneurship competition. Luckily we passed the preliminary round and semi-final round contests. In the competition, we delivered the precise synthetic biology concept to the audience and the judges. Let everyone knew how synthetic biology works.

8/1

Plasmid extraction and quantification:
GFP40(BBa_E0040), RBS33, terminator, CI, RBS30(BBa_B0030), FadL(cloned), FadR(cloned).
Redo whole genome extract from Bacillus licheniformis =>failed

8/2

Plasmid extraction: promoter J1 (J23101), promoter J0 (J23100)
Ligase the promoter part to our existed gene circuit part.
1. Cutting the promoter(J23100) with restriction enzymes SpeI, PstI, and cutting the previous gene circuits with XpaI and PatI,
2. Ligation the two parts overnights.
3. Transformation(overnight)
4. Colony PCR.

8/4

Ligation preparation: digestion and purification of CI and pCI =>pCI failed

8/5

Redo whole genome extract from Bacillus licheniformis =>failed
Redo digestion and purification of pCI =>failed again.
Possible reason:
1. After one week, the high-concentration pCI has degraded =>Redo measuring DNA concentration, but it didn’t decrease very much.
2. The high absorption at 260nm is actually contributed by RNA, not pCI. =>Add RNase, incubate for 15 minutes and measure again, but it didn’t decrease.
3. The high absorption at 260nm is actually contributed by other DNA, not pCI=>But there was no other band or even smear on the agarose gel.
We couldn’t think of other possible reason, so we decided to request a new one.

8/6

Redo plasmid extraction of pCI
Redo whole genome extract from Bacillus licheniformis =>low but acceptable concentration.

8/7

Digestion and purification of pCI =>failed again.

8/10

Redo bacterial culture of Bacillus licheniformis
PCR cloning of Bacillus licheniformis KerA gene=>succes

8/11

New pCI biobrick streaking

8/12

Redo whole genome extract from Bacillus licheniformis =>desirable concentration.
plasmid extraction of RBS34 =>failed
Digestion, purification, and ligation of RBS34-GFP, RBS34-FadL, and RBS34-FadR.
pCI-CI ligation and transformation =>the colonies were red, failed again.
Digestion and purification of Bacillus whole genome PCR product.

8/13

Plasmid extraction of new pCI, RBS30, CI-pCI, RBS34,
Digestion, purification, ligation and transformation of RBS34-GFP, RBS34-FadL,
RBS34-FadR, KerA-pSB1C3.

8/14

Digestion, purification, ligation and transformation of new CI-pCI.
Digestion, purification, and ligation of RBS34-GFP and RBS34-FadR

8/15

Plasmid extraction of FadL, KerA-pSB1C3, RBS33, GFP40
Digestion, purification, ligation and transformation of RBS34-CI
At that time, we assumed that we had assemble some gene circuits.
Some of them are successfully assembled, but others may be failure.
1. J23100 - RBS30 – FadR
2. J23100 - RBS30 – FadL
3. J23100 - RBS34 – FadR
4. J23100 - RBS34 – FadL
5. J23100 - RBS34 – GFP
6. J23100 - RBS30 – GFP
P.S：There arose a big problem! The promoter J23100 can’t assemble to our previous gene circuits, although we tried many times. We doubt that maybe the J23100in the stock is out of work. We failed many times, finally we decided to change the J23100 into J23119, hope this will work.

8/16

Plasmid extraction of pfadBA (BBa_K817002), CI-pCI, RBS33, terminator
Colony PCR of RBS34-GFP, RBS34-FadR

8/18

DNA electrophoresis confirming: KerA-pSB1C3, CI-pCI failed.
Colony PCR of CI-pCI and KerA-pSB1C3

8/19

Plasmid extraction of CI-pCI
Digestion, purification, ligation and transformation of KerA-pSB1C3

8/22

Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes
Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of FAD6 insert and pSB1C3 vector
Transform

8/23

Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1300bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight

8/24

Plasmid extraction
Submit for sequencing

8/25

GFP34 (BBa_E0034) plate streaking.
FAD6 sequence confirmed
Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes
Digestion of RBS-34 pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of FAD6 insert and RBS-34 pSB1C3 vector

8/27

Plasmid extraction of pSB1C3, RBS34-FadR, GFP34,

8/28

Digestion, purification, ligation and transformation of OmpA-pSB1C3, PelB-pSB1C3, PhoA-pSB1C3, (RBS34-FadR)-terminator, CI-pCI, GFP34-term, KerA-pSB1C3
Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1300bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight

8/29

Digestion, purification, ligation and transformation of GFP40-terminator, GFP34-terminator, RBS34-FadR-terminator, RBS34-GFP40-terminator, RBS34-FadL-terminator
Colony PCR of OmpA-pSB1C3, PelB-pSB1C3, PhoA-pSB1C3, 34-FadR-terminator, CI-pCI, GFP34-term, KerA-pSB1C3 =>success
Plasmid extraction
Digestion of RBS34-FAD6 with Xbal1 and Pst1 restriction enzymes
Digestion of BBa_B0015 terminator pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of RBS34-FAD6 insert and BBa_B0015 terminator pSB1C3 vector
Transform

8/30

Ligation of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI
Digestion of pfadBA
Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1400bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight

8/31

Transformation of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI
Plasmid extraction
Digestion of RBS34-FAD6-terminator with Xbal1 and Pst1 restriction enzymes
Digestion of PfadBa pSB1C3 vector and BBa_J23119 pSB1C3 with Spe1 and Pst1 restriction enzymes
Ligation of RBS34-FAD6-terminator insert and BBa_J23119 pSB1C3 vector
Ligation of RBS34-FAD6-terminator insert and PfadBa pSB1C3 vector
Transform

SEPTEMBER

9/1

Colony PCR of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI ---> succeed
Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1450bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight
mRNA extraction for the eighth time
Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana)
Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)
PCR(LOX1、HPL1 whole part without point mutation)
Gradient: 50°C~60°C
Result: Succeeded with HPL whole part at annealing temperature of 57°C but DNA band brightness is not intense enough. Failed with LOX1.
Gel extraction and purification (HPL1 whole part without point mutation)

9/2

Plasmid extraction of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI
Digestion of RBS34-CI-pCI、RBS30-CI-pCI
Ligation of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI
Plasmid extraction
PCR(LOX1、HPL1 whole part amplification)
Gradient: 50°C~60°C
Result: Succeeded with HPL whole part amplification at annealing temperature of 57°C. Failed with LOX1.
Gel extraction and purification (HPL1 whole part without point mutation)

9/3

Digestion of CI
Transformation of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI
Ligation of RBS34-CI
PCR(LOX1、HPL1 whole part amplification)
Gradient: 50°C~60°C
Result: Succeeded with HPL whole part amplification at annealing temperature of 57°C. Failed with LOX1.
Gel extraction and purification (HPL1 whole part without point mutation)

9/4

Colony PCR of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI ---> succeed
Transformation of RBS34-CI
PCR(HPL1 two point-mutated parts)
Gradient: 57°C
Result: Succeeded with both HPL1 point-mutated parts at annealing temperature of 57°C.
Gel extraction and purification (two HPL1 point-mutated parts )
Primer design: (LOX2 with two point mutated sites need to be done)

9/5

Plasmid extraction of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI、RBS34-CI
Digestion of RBS34-GFP40-terminator、pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI、RBS34-CI
Ligation of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator
PCR(HPL1 whole part with point mutation)
Gradient: 57°C
Result: Succeeded with HPL1 whole part with point mutation at annealing temperature of 57°C.
Gel extraction and purification (HPL1 whole part with point mutation)

9/7

Transformation of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator

9/10

Colony PCR of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator---> succeed

9/11

PCR(LOX2 whole part without point mutation)
Gradient: 50°C~60°C
Result: Failed
Digestion of HPL1 with Xbal1 and Pst1 restriction enzymes
Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of HPL1 insert and pSB1C3 vector
Transform

9/12

PCR(LOX2 whole part without point mutation)
Gradient: 45°C~65°C
Result: Failed
Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1100bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight

9/13

Plasmid extraction of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator
Plasmid extraction
Submit for sequencing

9/15

HPL1 sequence confirmed
Digestion of HPL1 with Xbal1 and Pst1 restriction enzyme
Digestion of RBS-34 pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of HPL1 insert and RBS-34 pSB1C3 vector
PCR(LOX2 three point mutated parts)
Gradient: 50°C~60°C
Result: Succeeded with LOX2 three point mutated parts at annealing temperature of 50°C~60°C.
Gel extraction and purification (LOX2 three point mutated parts)

9/16

Digestion of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator
Ligation of pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator

9/17

Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1200bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight
PCR(LOX2 for ligation of three point mutated parts: Ligation of first part and second part)
Gradient: 50°C~60°C
Result: Succeeded with Ligation of first part and second part at annealing temperature of 54°C and 57°C.
Gel extraction and purification (Ligation of first part and second part)

9/18

Plasmid extraction
Digestion of RBS34-HPL1 with Xbal1 and Pst1 restriction enzymes
Digestion of BBa_B0015 terminator pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of RBS34-HPL1 insert and BBa_B0015 terminator pSB1C3 vector
Transform
PCR(LOX2 whole part: Ligation of first-second part and third part)
Gradient: 57°C
Result: Failed

9/19

Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1200bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight

9/20

Digestion of J23119 promoter
Ligation of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator
Plasmid extraction
Digestion of RBS34-HPL1-terminator with Xbal1 and Pst1 restriction enzymes
Digestion of PfadBa pSB1C3 vector and BBa_J23119 pSB1C3 with Spe1 and Pst1 restriction enzymes
Ligation of RBS34-HPL1-terminator insert and BBa_J23119 pSB1C3 vector
Ligation of RBS34-FAD6-terminator insert and PfadBa pSB1C3 vector
Transform
PCR(LOX2 whole part: Ligation of first-second part and third part)
Gradient: 50°C~60°C
Result: Failed

9/21

Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1250bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight

9/22

Transformation of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator
Plasmid Extraction
PCR(LOX2 for ligation of three point mutated parts: Ligation of second part and third part)
Gradient: 50°C~60°C
Result: Succeeded with Ligation of second part and third part at annealing temperature of 57°C.
Gel extraction and purification (Ligation of second part and third part)

9/23

PCR(LOX2 whole part: Ligation of first part and second-third part)
Gradient: 57°C
Result: Failed

9/24

PCR(LOX2 whole part: Ligation of first part and second-third part)
Gradient: 50°C~60°C
Result: Failed

9/25

Colony PCR of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator、pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator ---> succeed
PCR(LOX2 : two parts, first part and second-third part without point mutation)
Gradient: 50°C~60°C
Result: Succeeded with LOX2 first part and second-third part without point mutation at annealing temperature of 57°C.
Gel extraction and purification (LOX2 first part and second-third part without point mutation)

9/27

Plasmid extraction of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator
Digestion of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator

9/28

PCR(LOX2 whole part: Ligation of first part and second-third part)
Gradient: 56°C~62°C
Result: Succeeded with Ligation of first part and second-third part at annealing temperature of 57°C.
Gel extraction and purification (LOX2 whole part with only one point mutation done. Still got one point mutation that need to be done)

9/29

Ligation of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator---> succeed
PCR(LOX2 whole part: Add three primers to achieve the point mutation left)
Gradient: 56°C~62°C
Result: Succeeded with the point mutation left and achieve the whole part at annealing temperature of 57°C.
Gel extraction and purification (LOX2 whole part with both mutation point done)
Digestion of LOX2 with Xbal1 and Pst1 restriction enzymes
Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of LOX2 insert and pSB1C3 vector
Transform

9/30

Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Failed

OCTOBER

10/1

Transformation of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator
Inoculate BBa_J23119-RBS34-FAD6-terminator and PfadBA-RBS34-FAD6-terminator into LB broth and cultivate under 37°C overnight

10/2

E.coli Lipid Extraction
Esterification
Gas chromatography
Result: Failed

10/3

Ligation of LOX2 insert and pSB1C3 vector
Transform

10/4

Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Failed

10/5

Colony PCR of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator

10/7

Plasmid extraction of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator、pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator

10/10

Testing the function of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator、pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator

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+       <a href="https://2014.igem.org/Team:NTU_Taida"><img id="logo" width="70px" src="https://static.igem.org/mediawiki/2014/1/19/NTU_Taida_logo.jpg"></a>
      </div>
@@ Line 77: / Line 78: @@
-       <li class="dropdown">
+ <li class="dropdown">
-           <a href="#" class="dropdown-toggle" data-toggle="dropdown">SAFETY<span class="caret"></span></a>
+           <a href="SAFETY" >SAFETY</a>
-          <ul class="dropdown-menu" role="menu">
+         </li>
-            <li><a href="SAFETY">What we done</a></li>
-            <li class="divider"></li>
-            <li><a href="SAFETY%20what%20else%20can%20we%20do">What else can we do</a></li>
-          </ul>
-        </li>
     <li class="dropdown">
@@ Line 100: / Line 95: @@
           </li>
+            <li class="dropdown">
+          <a href="#" class="dropdown-toggle" data-toggle="dropdown">SPONSORS<span class="caret"></span></a>
+          <ul class="dropdown-menu" role="menu">
+            <li><a href="Apex">ApexBio Biotech</a></li>
+            <li><a href="Lih">LihPao Life Science</a></li>
+          </ul>
+        </li>
          <li class="active">
@@ Line 105: / Line 109: @@
          MAIN PAGE 2014
          </a> </li>
+           <li class="active">
+        <a href="https://www.facebook.com/NationalTaiwanUniversityiGEM?fref=ts"><img src="https://static.igem.org/mediawiki/2014/5/5a/NTU_Taida_Facebook.jpg" width=100px;>
+        </a></li>
          </ul>
 </nav>
-   <div class="container">
+<div class="boxK"style="position:relative; top:100px;padding:200px;padding-top:25px;padding-bottom:25px">
+ <h2 style="background-color:#eeeeee">JUNE</h2>
+  <div style="padding-bottom:30px">
+  <h4>6/24</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+      mRNA extraction from Arabidopsis thaliana</p>
+Learn how to extract the plasmid.</p>
+Learn how to conduct the gel extraction and electrophoresis</p>
+Learn how to transform.</p>
+Learn how to do the gel extraction</p>
+Learn how to use the PCR Machine.</p>
+Design the primer for the FadL,R</p>
+</h5>
+  </div>
+    <div style="padding-bottom:30px">
+  <h4>6/25</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+RNA gel electrophoresis </p>
+Result: Failed (Smeared band indicated degradation of mRNA)</p>
+</h5>
+  </div>
+      <div style="padding-bottom:30px">
+  <h4>6/30</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+mRNA extraction for the second time</p>
+Result: Failed (Smeared band indicated degradation of mRNA)</p>
+Design the primer for the FadL,R</p>
+Determine whether to use the elongated primer(primer which contains the first 20 mer of FadR,L coding sequence and plus the restriction site which contains the E,X,S,P. </p>
+After heavily discussion, we decided to use the elongated primer. Though there is a big chance of failure, but it’s easy to operate.</p>
+Visit the 2014 Igem website and learn the rules set by the Igem.</p>
+Learn the biobrick design.</p>
+</h5>
+  </div>
+  <h2 style="background-color:#eeeeee">JULY</h2>
+         <div style="padding-bottom:30px">
+  <h4>7/1</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+mRNA extraction for the third time</p>
+Result: Failed (Smeared band indicated degradation of mRNA)</p>
+Use the primer we designed to get the FadR, L from E.coli.</p>
+Failed 4 times. But the fifth cloning experiment had a huge success.</p>
+Therefore, we have the Fad R/L, though they had already registered in the iGEM website(sample not in stock)</p>
+</h5>
+  </div>
+         <div style="padding-bottom:30px">
+  <h4>7/2</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+mRNA extraction for the fourth time</p>
+Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana) </p>
+Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)</p>
+Primer design: CCD1、LOX1、HPL1</p>
+HPL1 has to be point mutated in order to become suitable for IGEM backbone pSB1C3</p>
+</h5>
+  </div>
+            <div style="padding-bottom:30px">
+  <h4>7/15</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(CCD1、LOX1、HPL1)</p>
+Result: Failed</p>
+Using the biobrick, and transformed the biobrick plasmid to our DH5-alpha E.coli.</p>
+.	Ribosomal binding site: RBS30.RB34</p>
+.	Promoter: J23100, J23101</p>
+.	Terminator: B0015, B0012</p>
+.	Plasmid backbone: Psb1A3, Psb1C3, Psb1K3</p>
+.	GFP: E0034, E0040</p>
+Extract the plasmid and make them into stocks.</p>
+Design the PKEK sequence, with whole genome synthesis (But later this idea was disapproved by the professor an use the primer synthesis method)</p>
+Make discussion with dry lab member to determine which strategy should use for modeling our pathway. Later we all agree to use the GFP assay.</p>
+</h5>
+  </div>
+               <div style="padding-bottom:30px">
+  <h4>7/17</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(CCD1、LOX1、HPL1)</p>
+Gradient: 50°C~60°C</p>
+Result: Failed (Probably there are some problem with our cDNA)</p>
+Started to assemble the gene circuit. </p>
+.	The beginning of every circuit is to cut the RBS 30/34 with restriction enzymes SpeI & PstI. And the FadR/L is cut with XbaI and PstI restriction enzymes. </p>
+.	After digestion for four hours, we conducted the electrophoresis and gel extraction for our target DNA sequences.</p>
+.	We ligated the RBS30/34 and FadR/L with ligase.(ligation high2.0)</p>
+Overnight.</p>
+.	Transformed the overnight plasmid to the DH5alpha competent cell.</p>
+.	Colony PCR to make sure whether we had insert the right gene into our competent cells.</p>
+</h5>
+  </div>
+                  <div style="padding-bottom:30px">
+  <h4>7/18</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transformation of the following biobrick parts:</p>
+B0033 (RBS, weak),  C0051 (CI),  R0051 (pCI),  B0015 (terminator)</p>
+All of them using CP plate. </p>
+Results: R0051 (pCI) failed. </p>
+</h5>
+  </div>
+                     <div style="padding-bottom:30px">
+  <h4>7/19</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Bacterial culture: B0033 (RBS, weak),  C0051 (CI),  B0015 (terminator)</p>
+Redo R0051 (pCI) transformation =>failed again</p>
+Possible reason: We mistakenly took the plasmid backbone of R0051 as pSB1C3, which is actually pSB1A2, and therefore used the wrong antibiotics. On the next day, we used Amp+ plate, add more SOB in recovery, use another competent cell(XBL-blue), and did it again. </p>
+mRNA extraction for the fifth time</p>
+Result: Failed (Smeared band indicated degradation of mRNA)</p>
+mRNA extraction for the sixth time</p>
+Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana) </p>
+Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)</p>
+</h5>
+  </div>
+                        <div style="padding-bottom:30px">
+  <h4>7/20</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction: B0033 (RBS33, weak),  C0051 (CI),  B0015 (terminator)</p>
+(1、2 is terminator，3、4 is RBS， 5、6 is CI)</p>
+redo R0051 (pCI) transformation =>success!</p>
+PCR(CCD1、LOX1、HPL1)</p>
+Gradient: 50°C~60°C</p>
+Result: Succeeded with CCD1 at annealing temperature of 55°C, Failed with LOX1 and HPL1</p>
+Gel extraction and purification (CCD1)</p>
+</h5>
+  </div>
+                           <div style="padding-bottom:30px">
+  <h4>7/21</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Restriction enzyme digestion of RBS33, CI, and terminator, using PstI and EcoRI. </p>
+Then use DNA electrophoresis to confirm that all of them are correct. </p>
+bacterial culture of pCI. </p>
+PCR(LOX1、HPL1)</p>
+Result: Succeeded with one of HPL1’s point-mutated parts at annealing temperature of 52°C. Failed with LOX1 and HPL1’s another point-mutated parts.</p>
+Gel extraction and purification (one of HPL1’s point-mutated parts)</p>
+</h5>
+                              </div>
+                              <div style="padding-bottom:30px">
+  <h4>7/22</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of pCI and confirmation, using EcoRI digestion and DNA electrophoresis. The DNA concentration was too low that band was invisible on the agarose gel. </p>
+PCR(LOX1、HPL1)</p>
+Gradient: 50°C~60°C</p>
+Result: Failed with both LOX1 and HPL1</p>
+ Primer design: (FAD6、HPL1 redesign)</p>
+</h5>
+                                 </div>
+                                 <div style="padding-bottom:30px">
+  <h4>7/23</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo bacterial culture and plasmid extraction of pCI. Failed again.</p>
+Possible reason: We didn’t wait for a few minutes for MP2 to react, so the cell lysis step wasn’t complete. </p>
+Another project is to make the GFP assay. So we decided to ligate the RBS 34/30 with GFP. The method are identical to the Fad R/L gene circuits.</p>
+For now we have:</p>
+.	RBS30 – FadR</p>
+.	RBS30 – FadL</p>
+.	RBS34 – FadR</p>
+.	RBS34 – FadL</p>
+.	RBS34 – GFP</p>
+.	RBS30 – GFP</p>
+</h5>
+                                    </div>
+                                    <div style="padding-bottom:30px">
+  <h4>7/24</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(LOX1、HPL1’s another part)</p>
+Gradient: 45°C~65°C</p>
+Result: Failed with both LOX1 and HPL1</p>
+</h5>
+                                       </div>
+                                       <div style="padding-bottom:30px">
+  <h4>7/28</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo bacterial culture and plasmid extraction of pCI. </p>
+Redo transformation of pCI using another competent cell, DH5-alpha. </p>
+bacterial culture of Bacillus licheniformis at 30 ℃, using nutrient broth. </p>
+mRNA extraction for the seventh time</p>
+Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana) </p>
+Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)</p>
+</h5>
+                                          </div>
+                                          <div style="padding-bottom:30px">
+  <h4>7/29</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+plasmid extraction of pCI succeeded, but DNA electrophoresis failed, there was no band near 2.1k. Reason unknown. </p>
+Extract whole genome from Bacillus licheniformis. Failed. </p>
+Possible reason: </p>
+.	Bacillus licheniformis grows slower than E. coli. We didn’t give it enough time to grow. </p>
+.	Bacillus licheniformis is gram+, its thick cell wall may be harder to lyse.=> use another protocol.  </p>
+PCR(LOX1、HPL1’s another part)</p>
+Gradient: 50°C~60°C</p>
+Result: Failed with both LOX1 and HPL1</p>
+</h5>
+                                            </div>
+<div style="padding-bottom:30px">
+  <h4>7/30</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo bacterial culture of Bacillus licheniformis =>The bacteria didn’t grow. </p>
+Subculture of Bacillus licheniformis plate. =>success</p>
+Redo pCI DNA electrophoresis. There was an unclear band near 2.1k.  </p>
+PCR(FAD6)</p>
+Gradient: 50°C~60°C</p>
+Result: Succeeded with FAD6 at annealing temperature of 58°C</p>
+Gel extraction and purification (FAD6)</p>
+</h5>
+   </div>
+<div style="padding-bottom:30px">
+  <h4>7/31</h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo bacterial culture of Bacillus licheniformis =>The medium dried out because the cap didn’t fit.</p>
+Another project is to make the GFP assay. So we decided to ligate the RBS 34/30 with GFP. The method are identical to the Fad R/L gene circuits.</p>
+For now we have:</p>
+.	RBS30 – FadR</p>
+.	RBS30 – FadL</p>
+.	RBS34 – FadR</p>
+.	RBS34 – FadL</p>
+.	RBS34 – GFP</p>
+.	RBS30 – GFP</p>
+And we started to ligate the promoter part to the existing gene circuits.</p>
+Prepare the presentation and PPT for the 2014 Asia Igem conference. </p>
+The laboratory was polluted by a strange strain of E.coli, and it seems like this polluted bacteria also contained the RFP, which appears in our every electrophoresis result. But this strange phenomenon suddenly seized in the beginning of August</p>
+We decided to make our PKEK gene circuits by primer-annealing methods. And we ordered the primers and materials in the late July.</p>
+</h5>
+   </div>
+   <h2 style="background-color:#eeeeee">AUGUST</h2>
+    <h5 style="line-height:1.4em;font-size:110%;">
+    Attending the Igem Asia conference, exchanged our thought of signal sequence and PKEK. We got lots of inspiration and suggestion, which influenced our latter experiment methods and design.</p>
+We decided to bring our PKEK circuit in to commercial design. By fusion our PKEK circuits to the his-tag, we can order our E.coli to produce huge amounts of PKEK, and use the his-tag to catch our PKEK product. (This part is still under conistruction)</p>
+In order to make our Igem PKEK gene circuit more practical, we combined commercial competition with our PKEK project. We wanted to use the synthetic biology to produce huge amounts of medical peptides, which have high value and can make contribution to people all over the world. Therefore, we participated the China Innovation & Entrepreneurship competition. Luckily we passed the preliminary round and semi-final round contests. In the competition, we delivered the precise synthetic biology concept to the audience and the judges. Let everyone knew how synthetic biology works.</p>
+    </h5>
+                <div style="padding-bottom:30px">
+  <h4>8/1<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction and quantification:</p> GFP40(BBa_E0040), RBS33, terminator, CI, RBS30(BBa_B0030), FadL(cloned), FadR(cloned).</p>
+Redo whole genome extract from Bacillus licheniformis =>failed</p>
+</h5>
+   </div>
+                 <div style="padding-bottom:30px">
+  <h4>8/2<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction: promoter J1 (J23101), promoter J0 (J23100)</p>
+Ligase the promoter part to our existed gene circuit part.</p>
+.	Cutting the promoter(J23100) with restriction enzymes SpeI, PstI, and cutting the previous gene circuits with XpaI and PatI,</p>
+.	Ligation the two parts overnights.</p>
+.	Transformation(overnight)</p>
+.	Colony PCR.</p>
+</h5>
+   </div>
+                     <div style="padding-bottom:30px">
+  <h4>8/4<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Ligation preparation: digestion and purification of CI and pCI =>pCI failed
+</h5>
+   </div>
+                         <div style="padding-bottom:30px">
+  <h4>8/5<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo whole genome extract from Bacillus licheniformis =>failed</p>
+Redo digestion and purification of pCI =>failed again. </p>
+Possible reason:</p>
+.	After one week, the high-concentration pCI has degraded =>Redo measuring DNA concentration, but it didn’t decrease very much. </p>
+.	The high absorption at 260nm is actually contributed by RNA, not pCI. =>Add RNase, incubate for 15 minutes and measure again, but it didn’t decrease.</p>
+.	The high absorption at 260nm is actually contributed by other DNA, not pCI=>But there was no other band or even smear on the agarose gel. </p>
+We couldn’t think of other possible reason, so we decided to request a new one. </p>
+</h5>
+   </div>
+                             <div style="padding-bottom:30px">
+  <h4>8/6<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo plasmid extraction of pCI</p>
+Redo whole genome extract from Bacillus licheniformis =>low but acceptable concentration.</p>
+</h5>
+   </div>
+                                 <div style="padding-bottom:30px">
+  <h4>8/7<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion and purification of pCI =>failed again.
+</h5>
+   </div>
+                                     <div style="padding-bottom:30px">
+  <h4>8/10<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo bacterial culture of Bacillus licheniformis</P>
+PCR cloning of Bacillus licheniformis KerA gene=>succes</P>
+</h5>
+   </div>
+                                         <div style="padding-bottom:30px">
+  <h4>8/11<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+New pCI biobrick streaking
+</h5>
+   </div>
+                                             <div style="padding-bottom:30px">
+  <h4>8/12<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Redo whole genome extract from Bacillus licheniformis =>desirable concentration.</P>
+plasmid extraction of RBS34 =>failed</P>
+Digestion, purification, and ligation of RBS34-GFP, RBS34-FadL, and RBS34-FadR. </P>
+pCI-CI ligation and transformation =>the colonies were red, failed again. </P>
+Digestion and purification of Bacillus whole genome PCR product. </P>
+</h5>
+   </div>
+                                                 <div style="padding-bottom:30px">
+  <h4>8/13<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of new pCI, RBS30, CI-pCI, RBS34,  </P>
+Digestion, purification, ligation and transformation of RBS34-GFP, RBS34-FadL, </P>
+RBS34-FadR, KerA-pSB1C3. </P>
+</h5>
+   </div>
+                                                   <div style="padding-bottom:30px">
+  <h4>8/14<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion, purification, ligation and transformation of new CI-pCI.</P>
+Digestion, purification, and ligation of RBS34-GFP and RBS34-FadR</P>
+</h5>
+   </div>
+                                                       <div style="padding-bottom:30px">
+  <h4>8/15<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of FadL, KerA-pSB1C3, RBS33, GFP40</P>
+Digestion, purification, ligation and transformation of RBS34-CI</P>
+At that time, we assumed that we had assemble some gene circuits.</P>
+Some of them are successfully assembled, but others may be failure.</P>
+.	J23100 - RBS30 – FadR</P>
+.	J23100 - RBS30 – FadL</P>
+.	J23100 - RBS34 – FadR</P>
+.	J23100 - RBS34 – FadL</P>
+.	J23100 - RBS34 – GFP</P>
+.	J23100 - RBS30 – GFP</P>
+P.S：There arose a big problem! The promoter J23100 can’t assemble to our previous gene circuits, although we tried many times. We doubt that maybe the J23100in the stock is out of work. We failed many times, finally we decided to change the J23100 into J23119, hope this will work.</P>
+</h5>
+   </div>
+                                                           <div style="padding-bottom:30px">
+  <h4>8/16<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of pfadBA (BBa_K817002), CI-pCI, RBS33, terminator</P>
+Colony PCR of RBS34-GFP, RBS34-FadR</P>
+</h5>
+   </div>
+                                                               <div style="padding-bottom:30px">
+  <h4>8/18<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+DNA electrophoresis confirming: KerA-pSB1C3, CI-pCI failed. </P>
+Colony PCR of CI-pCI and KerA-pSB1C3</P>
+</h5>
+   </div>
+                                                                   <div style="padding-bottom:30px">
+  <h4>8/19<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of CI-pCI</P>
+Digestion, purification, ligation and transformation of KerA-pSB1C3</P>
+</h5>
+   </div>
+    <div style="padding-bottom:30px">
+  <h4>8/22<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes</P>
+Ligation of FAD6 insert and pSB1C3 vector</P>
+Transform</P>
+</h5>
+   </div>
+       <div style="padding-bottom:30px">
+  <h4>8/23<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1300bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+</h5>
+   </div>
+           <div style="padding-bottom:30px">
+  <h4>8/24<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction</P>
+Submit for sequencing</P>
+</h5>
+   </div>
+               <div style="padding-bottom:30px">
+  <h4>8/25<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+GFP34 (BBa_E0034) plate streaking.</P>
+FAD6 sequence confirmed</P>
+Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of RBS-34 pSB1C3 vector with Spe1 and Pst1 restriction enzymes</P>
+Ligation of FAD6 insert and RBS-34 pSB1C3 vector</P>
+</h5>
+   </div>
+                   <div style="padding-bottom:30px">
+  <h4>8/27<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of pSB1C3, RBS34-FadR, GFP34,
+</h5>
+   </div>
+                       <div style="padding-bottom:30px">
+  <h4>8/28<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion, purification, ligation and transformation of OmpA-pSB1C3, PelB-pSB1C3, PhoA-pSB1C3, (RBS34-FadR)-terminator, CI-pCI, GFP34-term, KerA-pSB1C3</P>
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1300bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+</h5>
+   </div>
+                           <div style="padding-bottom:30px">
+  <h4>8/29<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion, purification, ligation and transformation of GFP40-terminator, GFP34-terminator, RBS34-FadR-terminator, RBS34-GFP40-terminator, RBS34-FadL-terminator </P>
+Colony PCR of OmpA-pSB1C3, PelB-pSB1C3, PhoA-pSB1C3, 34-FadR-terminator, CI-pCI, GFP34-term, KerA-pSB1C3 =>success </P>
+Plasmid extraction</P>
+Digestion of RBS34-FAD6 with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of BBa_B0015 terminator pSB1C3 vector with Spe1 and Pst1 restriction enzymes</P>
+Ligation of RBS34-FAD6 insert and BBa_B0015 terminator pSB1C3 vector</P>
+Transform</P>
+</h5>
+   </div>
+                               <div style="padding-bottom:30px">
+  <h4>8/30<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Ligation of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI</P>
+Digestion of pfadBA</P>
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1400bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+</h5>
+   </div>
+                                   <div style="padding-bottom:30px">
+  <h4>8/31<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transformation of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI</P>
+Plasmid extraction</P>
+Digestion of RBS34-FAD6-terminator with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of PfadBa pSB1C3 vector and BBa_J23119 pSB1C3 with Spe1 and Pst1 restriction enzymes</P>
+Ligation of RBS34-FAD6-terminator insert and BBa_J23119 pSB1C3 vector</P>
+Ligation of RBS34-FAD6-terminator insert and PfadBa pSB1C3 vector</P>
+Transform</P>
+</h5>
+   </div>
+    <h2 style="background-color:#eeeeee">SEPTEMBER</h2>
+                                        <div style="padding-bottom:30px">
+  <h4>9/1<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Colony PCR of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI ---> succeed</P>
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1450bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+mRNA extraction for the eighth time</P>
+Result: Succeeded (Five Distinct bands indicate five chromosomes possessed by Arabidopsis thaliana) </P>
+Make cDNA from extracted mRNA using RNA dependent DNA reverse transcriptase (kit: Go script)</P>
+PCR(LOX1、HPL1 whole part without point mutation)</P>
+Gradient: 50°C~60°C</P>
+Result: Succeeded with HPL whole part at annealing temperature of 57°C but DNA band brightness is not intense enough. Failed with LOX1.</P>
+Gel extraction and purification (HPL1 whole part without point mutation)</P>
+</h5>
+   </div>
+                                             <div style="padding-bottom:30px">
+  <h4>9/2<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of RBS34-GFP40-terminator、RBS34-CI-pCI、RBS30-CI-pCI</P>
+Digestion of RBS34-CI-pCI、RBS30-CI-pCI</P>
+Ligation of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI</P>
+Plasmid extraction</P>
+PCR(LOX1、HPL1 whole part amplification)</P>
+Gradient: 50°C~60°C</P>
+Result: Succeeded with HPL whole part amplification at annealing temperature of 57°C. Failed with LOX1.</P>
+Gel extraction and purification (HPL1 whole part without point mutation)</P>
+</h5>
+   </div>
+<div style="padding-bottom:30px">
+  <h4>9/3<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion of CI</P>
+Transformation of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI</P>
+Ligation of RBS34-CI</P>
+PCR(LOX1、HPL1 whole part amplification)</P>
+Gradient: 50°C~60°C</P>
+Result: Succeeded with HPL whole part amplification at annealing temperature of 57°C. Failed with LOX1.</P>
+Gel extraction and purification (HPL1 whole part without point mutation)</P>
+</h5>
+   </div>
+     <div style="padding-bottom:30px">
+  <h4>9/4<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Colony PCR of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI  ---> succeed</P>
+Transformation of RBS34-CI</P>
+PCR(HPL1 two point-mutated parts)</P>
+Gradient: 57°C</P>
+Result: Succeeded with both HPL1 point-mutated parts at annealing temperature of 57°C.</P>
+Gel extraction and purification (two HPL1 point-mutated parts )</P>
+Primer design: (LOX2 with two point mutated sites need to be done)</P>
+</h5>
+   </div>
+          <div style="padding-bottom:30px">
+  <h4>9/5<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI、RBS34-CI</P>
+Digestion of RBS34-GFP40-terminator、pfadBA-RBS34-CI-pCI、pfadBA-RBS30-CI-pCI、RBS34-CI</P>
+Ligation of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator</P>
+PCR(HPL1 whole part with point mutation)</P>
+Gradient: 57°C</P>
+Result: Succeeded with HPL1 whole part with point mutation at annealing temperature of 57°C.</P>
+Gel extraction and purification (HPL1 whole part with point mutation)</P>
+</h5>
+   </div>
+               <div style="padding-bottom:30px">
+  <h4>9/7<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transformation of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator
+</h5>
+   </div>
+                    <div style="padding-bottom:30px">
+  <h4>9/10<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Colony PCR of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator---> succeed
+</h5>
+   </div>
+                         <div style="padding-bottom:30px">
+  <h4>9/11<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(LOX2 whole part without point mutation)</P>
+Gradient: 50°C~60°C</P>
+Result: Failed</P>
+Digestion of HPL1 with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes</P>
+Ligation of HPL1 insert and pSB1C3 vector</P>
+Transform</P>
+</h5>
+   </div>
+                              <div style="padding-bottom:30px">
+  <h4>9/12<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(LOX2 whole part without point mutation)</P>
+Gradient: 45°C~65°C</P>
+Result: Failed</P>
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1100bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+</h5>
+   </div>
+                                   <div style="padding-bottom:30px">
+  <h4>9/13<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator</P>
+Plasmid extraction</P>
+Submit for sequencing</P>
+</h5>
+   </div>
+                                        <div style="padding-bottom:30px">
+  <h4>9/15<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+HPL1 sequence confirmed</P>
+Digestion of HPL1 with Xbal1 and Pst1 restriction enzyme</P>
+Digestion of RBS-34 pSB1C3 vector with Spe1 and Pst1 restriction enzymes</P>
+Ligation of HPL1 insert and RBS-34 pSB1C3 vector</P>
+PCR(LOX2 three point mutated parts)</P>
+Gradient: 50°C~60°C</P>
+Result: Succeeded with LOX2 three point mutated parts at annealing temperature of 50°C~60°C.</P>
+Gel extraction and purification (LOX2 three point mutated parts)</P>
+</h5>
+   </div>
+                                             <div style="padding-bottom:30px">
+  <h4>9/16<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator</P>
+Ligation of pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator</P>
+</h5>
+   </div>
+                                                  <div style="padding-bottom:30px">
+  <h4>9/17<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1200bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+PCR(LOX2 for ligation of three point mutated parts: Ligation of first part and second part)</P>
+Gradient: 50°C~60°C</P>
+Result: Succeeded with Ligation of first part and second part at annealing temperature of 54°C and 57°C.</P>
+Gel extraction and purification (Ligation of first part and second part)</P>
+</h5>
+   </div>
+                                                       <div style="padding-bottom:30px">
+  <h4>9/18<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction</P>
+Digestion of RBS34-HPL1 with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of BBa_B0015 terminator pSB1C3 vector with Spe1 and Pst1 restriction enzymes</P>
+Ligation of RBS34-HPL1 insert and BBa_B0015 terminator pSB1C3 vector</P>
+Transform</P>
+PCR(LOX2 whole part: Ligation of first-second part and third part)</P>
+Gradient: 57°C</P>
+Result: Failed</P>
+</h5>
+   </div>
+                                                            <div style="padding-bottom:30px">
+  <h4>9/19<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1200bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+</h5>
+   </div>
+                                                                 <div style="padding-bottom:30px">
+  <h4>9/20<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Digestion of J23119 promoter</P>
+Ligation of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator</P>
+Plasmid extraction</P>
+Digestion of RBS34-HPL1-terminator with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of PfadBa pSB1C3 vector and BBa_J23119 pSB1C3 with Spe1 and Pst1 restriction enzymes</P>
+Ligation of RBS34-HPL1-terminator insert and BBa_J23119 pSB1C3 vector</P>
+Ligation of RBS34-FAD6-terminator insert and PfadBa pSB1C3 vector</P>
+Transform</P>
+PCR(LOX2 whole part: Ligation of first-second part and third part)</P>
+Gradient: 50°C~60°C</P>
+Result: Failed</P>
+</h5>
+   </div>
+<div style="padding-bottom:30px">
+  <h4>9/21<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+	Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Succeeded( with band at around 1250bp)</P>
+Inoculate valid colony into LB broth and cultivate under 37°C overnight</P>
+</h5>
+   </div>
+     <div style="padding-bottom:30px">
+  <h4>9/22<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transformation of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator</P>
+Plasmid Extraction</P>
+PCR(LOX2 for ligation of three point mutated parts: Ligation of second part and third part)</P>
+Gradient: 50°C~60°C</P>
+Result: Succeeded with Ligation of second part and third part at annealing temperature of 57°C.</P>
+Gel extraction and purification (Ligation of second part and third part)</P>
+</h5>
+   </div>
+          <div style="padding-bottom:30px">
+  <h4>9/23<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(LOX2 whole part: Ligation of first part and second-third part)</P>
+Gradient: 57°C</P>
+Result: Failed</P>
+</h5>
+   </div>
+               <div style="padding-bottom:30px">
+  <h4>9/24<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(LOX2 whole part: Ligation of first part and second-third part)</P>
+Gradient: 50°C~60°C</P>
+Result: Failed</P>
+</h5>
+   </div>
+                    <div style="padding-bottom:30px">
+  <h4>9/25<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Colony PCR of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator、pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator ---> succeed</P>
+PCR(LOX2 : two parts, first part and second-third part without point mutation)</P>
+Gradient: 50°C~60°C </P>
+Result: Succeeded with LOX2 first part and second-third part without point mutation at annealing temperature of 57°C.</P>
+Gel extraction and purification (LOX2 first part and second-third part without point mutation)</P>
+</h5>
+   </div>
+                         <div style="padding-bottom:30px">
+  <h4>9/27<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator</P>
+Digestion of J23119-RBS34-FadR-terminator、J23119-RBS34-FadL-terminator</P>
+</h5>
+   </div>
+                              <div style="padding-bottom:30px">
+  <h4>9/28<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+PCR(LOX2 whole part: Ligation of first part and second-third part)</P>
+Gradient: 56°C~62°C</P>
+Result: Succeeded with Ligation of first part and second-third part at annealing temperature of 57°C.</P>
+Gel extraction and purification (LOX2 whole part with only one point mutation done. Still got one point mutation that need to be done)</P>
+</h5>
+   </div>
+                                   <div style="padding-bottom:30px">
+  <h4>9/29<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Ligation of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator---> succeed</P>
+PCR(LOX2 whole part: Add three primers to achieve the point mutation left)</P>
+Gradient: 56°C~62°C</P>
+Result: Succeeded with the point mutation left and achieve the whole part at annealing temperature of 57°C.</P>
+Gel extraction and purification (LOX2 whole part with both mutation point done)</P>
+Digestion of LOX2 with Xbal1 and Pst1 restriction enzymes</P>
+Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes</P>
+Ligation of LOX2 insert and pSB1C3 vector</P>
+Transform</P>
+</h5>
+   </div>
+                                        <div style="padding-bottom:30px">
+  <h4>9/30<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Failed</P>
+</h5>
+   </div>
+     <h2 style="background-color:#eeeeee">OCTOBER</h2>
+ <div style="padding-bottom:30px">
+  <h4>10/1<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transformation of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator</P>
+Inoculate BBa_J23119-RBS34-FAD6-terminator and PfadBA-RBS34-FAD6-terminator into LB broth and cultivate under 37°C overnight</P>
+</h5>
+   </div>
+       <div style="padding-bottom:30px">
+  <h4>10/2<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+E.coli Lipid Extraction</P>
+Esterification</P>
+Gas chromatography</P>
+Result: Failed</P>
+</h5>
+   </div>
+             <div style="padding-bottom:30px">
+  <h4>10/3<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Ligation of LOX2 insert and pSB1C3 vector</P>
+Transform</P>
+</h5>
+   </div>
+                   <div style="padding-bottom:30px">
+  <h4>10/4<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Transform result: succeeded with colonies form on CP resistant plate</P>
+Colony PCR (To check whether the colonies are valid)</P>
+Result: Failed</P>
+</h5>
+   </div>
+                         <div style="padding-bottom:30px">
+  <h4>10/5<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Colony PCR of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator
+</h5>
+   </div>
+                               <div style="padding-bottom:30px">
+  <h4>10/7<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Plasmid extraction of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator、pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator
+</h5>
+   </div>
+                                     <div style="padding-bottom:30px">
+  <h4>10/10<h4>
+  <h5 style="line-height:1.4em;font-size:110%;">
+Testing the function of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator、pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator
+</h5>
+   </div>
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Team:NTU Taida/LOGBOOK

From 2014.igem.org

Latest revision as of 01:10, 18 October 2014

JUNE

6/24

mRNA extraction from Arabidopsis thaliana Learn how to extract the plasmid. Learn how to conduct the gel extraction and electrophoresis Learn how to transform. Learn how to do the gel extraction Learn how to use the PCR Machine. Design the primer for the FadL,R

6/25

RNA gel electrophoresis Result: Failed (Smeared band indicated degradation of mRNA)

6/30

JULY

7/1

7/2

7/15

7/17

7/18

Transformation of the following biobrick parts: B0033 (RBS, weak), C0051 (CI), R0051 (pCI), B0015 (terminator) All of them using CP plate. Results: R0051 (pCI) failed.

7/19

7/20

7/21

7/22

Plasmid extraction of pCI and confirmation, using EcoRI digestion and DNA electrophoresis. The DNA concentration was too low that band was invisible on the agarose gel. PCR(LOX1、HPL1) Gradient: 50°C~60°C Result: Failed with both LOX1 and HPL1 Primer design: (FAD6、HPL1 redesign)

7/23

7/24

PCR(LOX1、HPL1’s another part) Gradient: 45°C~65°C Result: Failed with both LOX1 and HPL1

7/28

7/29

7/30

7/31

AUGUST

8/1

Plasmid extraction and quantification: GFP40(BBa_E0040), RBS33, terminator, CI, RBS30(BBa_B0030), FadL(cloned), FadR(cloned). Redo whole genome extract from Bacillus licheniformis =>failed

8/2

8/4

Ligation preparation: digestion and purification of CI and pCI =>pCI failed

8/5

8/6

Redo plasmid extraction of pCI Redo whole genome extract from Bacillus licheniformis =>low but acceptable concentration.

8/7

Digestion and purification of pCI =>failed again.

8/10

Redo bacterial culture of Bacillus licheniformis PCR cloning of Bacillus licheniformis KerA gene=>succes

8/11

New pCI biobrick streaking

8/12

8/13

Plasmid extraction of new pCI, RBS30, CI-pCI, RBS34, Digestion, purification, ligation and transformation of RBS34-GFP, RBS34-FadL, RBS34-FadR, KerA-pSB1C3.

8/14

Digestion, purification, ligation and transformation of new CI-pCI. Digestion, purification, and ligation of RBS34-GFP and RBS34-FadR

8/15

8/16

Plasmid extraction of pfadBA (BBa_K817002), CI-pCI, RBS33, terminator Colony PCR of RBS34-GFP, RBS34-FadR

8/18

DNA electrophoresis confirming: KerA-pSB1C3, CI-pCI failed. Colony PCR of CI-pCI and KerA-pSB1C3

8/19

Plasmid extraction of CI-pCI Digestion, purification, ligation and transformation of KerA-pSB1C3

8/22

Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes Ligation of FAD6 insert and pSB1C3 vector Transform

8/23

Transform result: succeeded with colonies form on CP resistant plate Colony PCR (To check whether the colonies are valid) Result: Succeeded( with band at around 1300bp) Inoculate valid colony into LB broth and cultivate under 37°C overnight

8/24

Plasmid extraction Submit for sequencing

8/25

GFP34 (BBa_E0034) plate streaking. FAD6 sequence confirmed Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes Digestion of RBS-34 pSB1C3 vector with Spe1 and Pst1 restriction enzymes Ligation of FAD6 insert and RBS-34 pSB1C3 vector

8/27

Plasmid extraction of pSB1C3, RBS34-FadR, GFP34,

8/28

8/29

8/30

8/31

SEPTEMBER

9/1

9/2

9/3

9/4

9/5

9/7

Transformation of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator

9/10

Colony PCR of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator---> succeed

9/11

mRNA extraction from Arabidopsis thaliana
Learn how to extract the plasmid.
Learn how to conduct the gel extraction and electrophoresis
Learn how to transform.
Learn how to do the gel extraction
Learn how to use the PCR Machine.
Design the primer for the FadL,R

RNA gel electrophoresis
Result: Failed (Smeared band indicated degradation of mRNA)

Transformation of the following biobrick parts:
B0033 (RBS, weak), C0051 (CI), R0051 (pCI), B0015 (terminator)
All of them using CP plate.
Results: R0051 (pCI) failed.

Plasmid extraction of pCI and confirmation, using EcoRI digestion and DNA electrophoresis. The DNA concentration was too low that band was invisible on the agarose gel.
PCR(LOX1、HPL1)
Gradient: 50°C~60°C
Result: Failed with both LOX1 and HPL1
Primer design: (FAD6、HPL1 redesign)

PCR(LOX1、HPL1’s another part)
Gradient: 45°C~65°C
Result: Failed with both LOX1 and HPL1

Plasmid extraction and quantification:
GFP40(BBa_E0040), RBS33, terminator, CI, RBS30(BBa_B0030), FadL(cloned), FadR(cloned).
Redo whole genome extract from Bacillus licheniformis =>failed

Redo plasmid extraction of pCI
Redo whole genome extract from Bacillus licheniformis =>low but acceptable concentration.

Redo bacterial culture of Bacillus licheniformis
PCR cloning of Bacillus licheniformis KerA gene=>succes

Plasmid extraction of new pCI, RBS30, CI-pCI, RBS34,
Digestion, purification, ligation and transformation of RBS34-GFP, RBS34-FadL,
RBS34-FadR, KerA-pSB1C3.

Digestion, purification, ligation and transformation of new CI-pCI.
Digestion, purification, and ligation of RBS34-GFP and RBS34-FadR

Plasmid extraction of pfadBA (BBa_K817002), CI-pCI, RBS33, terminator
Colony PCR of RBS34-GFP, RBS34-FadR

DNA electrophoresis confirming: KerA-pSB1C3, CI-pCI failed.
Colony PCR of CI-pCI and KerA-pSB1C3

Plasmid extraction of CI-pCI
Digestion, purification, ligation and transformation of KerA-pSB1C3

Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes
Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of FAD6 insert and pSB1C3 vector
Transform

Transform result: succeeded with colonies form on CP resistant plate
Colony PCR (To check whether the colonies are valid)
Result: Succeeded( with band at around 1300bp)
Inoculate valid colony into LB broth and cultivate under 37°C overnight

Plasmid extraction
Submit for sequencing

GFP34 (BBa_E0034) plate streaking.
FAD6 sequence confirmed
Digestion of FAD6 with Xbal1 and Pst1 restriction enzymes
Digestion of RBS-34 pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of FAD6 insert and RBS-34 pSB1C3 vector

PCR(LOX2 whole part without point mutation)
Gradient: 50°C~60°C
Result: Failed
Digestion of HPL1 with Xbal1 and Pst1 restriction enzymes
Digestion of pSB1C3 vector with Spe1 and Pst1 restriction enzymes
Ligation of HPL1 insert and pSB1C3 vector
Transform

Plasmid extraction of pfadBA-RBS34-CI、pfadBA-RBS34-CI-pCI- RBS34-GFP40-terminator、pfadBA-RBS30-CI-pCI- RBS34-GFP40-terminator
Plasmid extraction
Submit for sequencing