Team:NTU Taida/LOGBOOK

From 2014.igem.org

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