iGEM 2022 — Team ccu-taiwan

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MAY.

JUN.

JUL.

AUG.

SEP.

OCT.

WET LAB

DRY LAB

HUMAN PRACTICE

# NOTEBOOK

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![](https://static.igem.wiki/teams/4131/wiki/drop.png)WET LAB

![](https://static.igem.wiki/teams/4131/wiki/drop.png)DRY LAB

![](https://static.igem.wiki/teams/4131/wiki/drop.png)HUMAN PRACTICE

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solution for CVD

Learn

Explore

Design

Expression promoter

Learn and Explore

Design

Redesign necessary

EPA?

Explore

Build

# From docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA)

# Identify the problem - solution for CVD

  According to the official statistics from the World Health Organization (WHO), cardiovascular
disease (CVD) is the leading cause of death and disability worldwide, causing 17.9 million deaths per year.
Statistics in Taiwan also indicate the seriousness of CVD locally.

  One important cause of CVD is the accumulation of oxidized low-density lipoprotein (LDL) in
blood vessels. Taking Omega-3 polyunsaturated fatty acid (PUFA) supplements, including DHA and EPA, could
effectively slow CVD progression.

![](https://static.igem.wiki/teams/4131/wiki/engineering/engineering01.png)

## Learn

  The human body can synthesize DHA from α-linolenic acid (ALA), which is abundant in some plant
oils. However, the conversion rate is extremely low, reported to be from 2-10% (Chiu, Su et al. 2008) or
even as low as 0.01% (Hussein, Ah-Sing et al. 2005). Therefore, DHA-rich foods, such as fish, or DHA
supplements are the two main sources for humans to get enough DHA. Notably, most DHA supplements are also
purified from fish, suggesting that supply of supplemental DHA will become a problem as the marine resources
are exhausted.

![](https://static.igem.wiki/teams/4131/wiki/engineering/engineering02.png)

## Explore

  Since fish is almost the only source of DHA, we attempted to figure out if we could generate an
alternative source of DHA by synthetic biology. Accordingly, we first found that oily fish, such as salmon
or sardines, do not generate DHA by themself. Instead, these fish uptake DHA-rich algae and accumulate DHA
in the body. Exploration of DHA-generating algae and other organisms showed that the deep-sea bacteria
*Moritella marina* MP-1 may be the excellent target for the following reasons (Yazawa 1996):

a. The genome sequence of MP-1 is known, suggesting that we have a chance to build the biobricks for DHA
production.

b. The *pfa* genes responsible for DHA production are known (Okuyama, Orikasa et al. 2007).

c. It had been shown that the ectopic expression of *pfa* genes in *E. coli* induced DHA
generation (Orikasa, Nishida et al. 2006).

## Design

  Following the research reported by Orisaka et. al., we extracted the *pfa* gene sequences
from MP-1 genome, and found that the five *pfa* genes, *pfa A*, *pfa B*, *pfa C*,
*pfa D* and *pfa E* are 7959 bp, 2652bp, 6036bp, 1617bp and 864bp in length, respectively (Orikasa,
Nishida et al. 2006). Because of the length limitation of each plasmid, we decided to clone these *pfa*
genes into different vectors. To balance the final length of each vector, we divided the *pfa* genes
into two groups. The *pfa A* and *pfa D* belong to group 1 (9576bp in total) and others belong to
group 2 (9552bp in total). Because gene expression in prokaryotes is poly-cistronic, we inserted a ribosome
binding site before each gene to regulate the protein translation.

![](https://static.igem.wiki/teams/4131/wiki/engineering/engineering03.png)

# Identify the the problem - expression promoter

  While deciding a promoter for protein expression, it is common to select the lac-induced
promoter to induce protein expression through lactose analog supplement. However, due to the extraordinary
size of the *pfa A* gene, it is highly possible that expression would form inclusion bodies and thus
loss of activity.

## Learn and Explore

  Since MP-1 is a deep-sea bacteria grown at low temperature, and low temperature promotes the
proper folding of large proteins (i.e. *pfa A*), we decided to apply the cold-inducible promoter cspA
for *pfa A* expression (Vasina and Baneyx 1997).

## Design

  Accordingly, we designed the *pfa A* and *pfa D* genes under the control of the CspA
promoter, while *pfa B'*, *pfa C* and *pfa E* genes are under the control of the lac
inducible promoter.

![](https://static.igem.wiki/teams/4131/wiki/engineering/engineering04.png)

## Redesign necessary

  After we constructed the plasmid design, we consulted different experts for suggestions. The
secondary PI, Dr. Lee, admired the cspA promoter designs and gave some suggestions for dealing with protein
aggregation. However, unexpectedly, another adviser, Dr. G. Rau, sent some references showing that DHA and
EPA had different effects in CVD prevention (Mason, Sherratt et al. 2022, Sherratt, Libby et al. 2022).
Further consultation with a cardiologist (name not shown, by request) from National Taiwan University
Hospital supported the opinion that EPA, but not DHA, is functional in CVD prevention .

# Identify the problem - EPA?

  EPA can prevent CVDs in different ways. In arteries, EPA preserves the membrane structure and
regulates cholesterol distribution. In blood cells, EPA can split heme into several reducers to remove the
free radicals, and reduce oxidative LDL accumulation. Consequently, EPA prevents the inflammation caused by
oxidative LDL. In the vascular environment, EPA can also relax the surrounding smooth muscle by enhancing
nitric oxide production to avoid severe CVDs (Peter, John et al. 2022).

## Explore

  To switch from DHA to EPA production without dramatically changing the whole design, we turned
back to survey references indicating that the DHA to EPA switch is feasible. Fortunately, we found that
*pfa B* is responsible for the final product formation. Importantly, the swapping of *pfa B* from
*Moritella marina* with *pfa B'* from *Shewanella pneumatophori* could shift the product from
DHA to EPA (Orikasa, Tanaka et al. 2009).

## Build

  Finally, we exchanged the *Moritella marina* *pfa B* with *Shewanella
pneumatophori* *pfa B'* and established a switchable PUFA generation system.

Reference:

1. Chiu, C. C., K. P. Su, T. C. Cheng, H. C. Liu, C. J. Chang, M. E. Dewey, R. Stewart and S. Y. Huang
   (2008). "The effects of omega-3 fatty acids monotherapy in Alzheimer's disease and mild cognitive
   impairment: a preliminary randomized double-blind placebo-controlled study." Prog Neuropsychopharmacol
   Biol Psychiatry 32(6): 1538-1544
2. Hussein, N., E. Ah-Sing, P. Wilkinson, C. Leach, B. A. Griffin and D. J. Millward (2005). "Long-chain
   conversion of [13C]linoleic acid and alpha-linolenic acid in response to marked changes in their dietary
   intake in men." J Lipid Res 46(2): 269-280.
3. Mason, R. P., S. C. R. Sherratt and R. H. Eckel (2022). "Omega-3-fatty acids: Do they prevent
   cardiovascular disease?" Best Pract Res Clin Endocrinol Metab: 101681.
4. Okuyama, H., Y. Orikasa, T. Nishida, K. Watanabe and N. Morita (2007). "Bacterial genes responsible for
   the biosynthesis of eicosapentaenoic and docosahexaenoic acids and their heterologous expression." Appl
   Environ Microbiol 73(3): 665-670.
5. Orikasa, Y., T. Nishida, A. Yamada, R. Yu, K. Watanabe, A. Hase, N. Morita and H. Okuyama (2006).
   "Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli."
   Biotechnol Lett 28(22): 1841-1847.
6. Orikasa, Y., M. Tanaka, S. Sugihara, R. Hori, T. Nishida, A. Ueno, N. Morita, Y. Yano, K. Yamamoto, A.
   Shibahara, H. Hayashi, Y. Yamada, A. Yamada, R. Yu, K. Watanabe and H. Okuyama (2009). "pfaB products
   determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis." FEMS
   Microbiol Lett 295(2): 170-176.
7. Peter, P. Toth, M. J. Chapman, K. G.Parhofer, J. R. Nelsone (2022). "Differentiating EPA from EPA/DHA in
   cardiovascular risk reduction." American Heart Journal Plus: Cardiology Research and Practice 17:100148
8. Sherratt, S. C. R., P. Libby, D. L. Bhatt and R. P. Mason (2022). "A biological rationale for the
   disparate effects of omega-3 fatty acids on cardiovascular disease outcomes." Prostaglandins Leukot
   Essent Fatty Acids 182: 102450.
9. Vasina, J. A. and F. Baneyx (1997). "Expression of aggregation-prone recombinant proteins at low
   temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems." Protein Expr
   Purif 9(2): 211-218.
10. Yazawa, K. (1996). "Production of eicosapentaenoic acid from marine bacteria." Lipids 31 Suppl:
    S297-300.

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Cloning pColdI-*pfa A*-*pfa D*

Cloning pSTV28-*pfa B*-*pfa C*-*pfa E*

Cloning pET28a-*AccBC*-*AccD1E*

Triple antibiotics selection

# Cloning pColdI-*pfa A*-*pfa D*

# Major experiment:

1. Amplifying the *pfa A* and *pfa D* genes from the genome of *Moritella
   marina*.
2. Cloning *pfa A* and *pfa D* genes into pColdI expression vector.
3. Expressing the pfa A and pfa D protein.

# Achievements:

Successfully amplifying the *pfa A* and *pfa D* genes, cloning *pfa D*
into pColdI vector and expressing pfa D protein.

# Associated result:

## Confirming the digested pColdI plasmids

  The pColdI plasmids were extracted from the cultured bacteria and subjected to NdeI and
XbaI digestion. The gel electrophoresis result showed the backbone of pColdI (4.3 kb) and stuffer (1.3
kb) as expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result01.png)

## Amplifying *pfa A* by PCR

  To clone the extraordinarily large (~8 kb) *pfa A* from the genome of *Moritella
marina*, we designed primers to clone two regions, *pfa A1* (~4.8 kb) and *pfa A2* (~4 kb). The *pfa A1* and *pfa A2* can be fused into the full
length *pfa A* though an endogenous EcoRI site.

  The restriction enzyme NdeI site at the 5' terminal of *pfa A1* was generated with the
forward primer and added through PCR using pfu DNA polymerase.

  The primer sequence are listed below:

  NdeI-*pfa A1*-F: CATATGGCTAAAAAGAACACCACATC

*pfa A1*-R: CCGCGGAGGTAAGTCAGTAA

  The gel electrophoresis result showed the size of *pfa A1* amplicon (4.8 kb) as
expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result02.png)Figure 2: The agarose image shows the amplicon of *pfa A1*.

  The restriction enzyme SacII site at 3' terminal of *pfa A2* was generated with the
reverse primer pair and added through PCR using pfu DNA polymerase.

  The primer sequences are listed below:

*pfa A2* 386-F: TGACTTAGGTATCGACTCAA

  SacII-*pfa A2*-R: CCGCGGTTATGACATATCGTTCAAAAT

  The gel electrophoresis result showed the size of *pfa A2* amplicon (4 kb) as expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result03.png)Figure 3: The agarose image shows the amplicon of *pfa A2*.

  In addition, we also tried to amplify the whole *pfa A* without splitting. According, we
performed PCR with the following primer pairs with using pfu DNA polymerase:

  NdeI-*pfa A1*-F: CATATGGCTAAAAAGAACACCACATC

  SacII-*pfa A2*-R: CCGCGGTTATGACATATCGTTCAAAAT

  The gel electrophoresis result showed the size of *pfa A* amplicon (8 kb) as expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result04.png)Figure 4: The agarose image shows the amplicon of *pfa A*.

## Amplifying *pfa D* by PCR

  To clone *pfa D* (~1.6 kb) from the genome of *Moritella marina*, we designed
a forward primer with a ribosome binding site (RBS) and a restriction enzyme SacII site at the 5'
terminal, while the 3' terminal of reverse primer is ended by restriction enzyme XbaI site. PCR
amplification was performed with the designed primer pair and pfu DNA polymerase.

  The primer sequences are listed below:

  SacII-RBS-*pfa D*-F:

  CCGCGGAGATATACCATGTCGAGTTTAGGTTTTAA

  XbaI-*pfa D*-R: TCTAGATTAATCACTCGTACGATAACTTG

  The gel electrophoresis result showed the size of *pfa D* amplicon (1.6 kb) as
expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result05.png)Figure 5: The agarose image shows the amplicon of *pfa D*.

## The TA cloning of *pfa D* and sequencing confirmation

  After gel electrophoresis, the *pfa D* amplicon was purified from agarose gel and
ligated with TA-vector. The ligated TA-*pfa D* was then transformed into *E. coli* DH5α
strain, and the colonies after antibiotic (Amp) selection were subjected to colony PCR.

![](https://static.igem.wiki/teams/4131/wiki/result/result06.png)Figure 6: The agarose image shows the colony PCR result of
TA-*pfa D*.

  The clones showing expected amplicon size (1.6 kb) in colony PCR were then expanded for
TA-*pfa D* plasmid extraction, and the extracted TA-*pfa D* plasmids were subjected to
restriction enzyme digestion by HindIII.

![](https://static.igem.wiki/teams/4131/wiki/result/result07.png)Figure 7: The agarose image shows the digestion result of
TA-*pfa D*.

  The clones showing expected amplicon size (1.6 kb) in colony PCR were then expanded for
TA-*pfa D* plasmid extraction, and the extracted TA-*pfa D* plasmids were subjected to
restriction enzyme digestion by HindIII.

![](https://static.igem.wiki/teams/4131/wiki/result/result08.png)Figure 8: The electrogram shows the Sanger sequencing result of
TA-*pfa D*.

  Finally, we sequence-confirmed the TA-*pfa D*.

## The cloning of *pfa D* into pColdI vector.

  The *pfa D* amplicon is excised from the TA-*pfa D* vector by SacII and XbaI
digestion, and the purified amplicon is ligated into pColdI vector. The colonies grown after standard
transformation were examined by colony PCR using a primer pair recognizing *pfa D* 1369-F and
pColdI-R. The size of the target amplicon of colony PCR is 0.3 kb in length.

  The primer sequences are listed below:

*pfa D* 1369-F: TCTTCACGCTGGTCAAACAC

  pColdI-R: CCAAATGGCAGGGATCTTAG

![](https://static.igem.wiki/teams/4131/wiki/result/result09.png)Figure 9: The agarose image shows the colony PCR result of
pColdI-*pfa D*.

  The pColdI-*pfa D* vectors were then purified from colonies and confirmed by Sanger
sequencing.

![](https://static.igem.wiki/teams/4131/wiki/result/result10.png)Figure 10: The electrogram shows the Sanger sequencing result of
pColdI-*pfa D*.

## The protein expression of pfa D

  We transformed pColdI-*pfa D* into *E. coli* BL21 strain and induced protein
expression through culturing bacteria in TB medium with 1 mM IPTG at 16 °C for 4 hr. The bacteria was
then harvested and subjected to SDS-PAGE analysis and Coomassie blue staining. The predicted size of
pfa D protein is 60 kDa. However, we did not observe the induced protein at the corresponding size. We
inferred that the induction was inefficient.

![](https://static.igem.wiki/teams/4131/wiki/result/result11.png)Figure 11: The Coomassie blue staining showed the induction of
pfa D proteins.

  Therefore, we extended the induction time to 9, 11 and 13 hr. All collected samples were
subjected to SDS-PAGE analysis and Coomassie blue staining. In the staining image, we clearly observed
the induced protein with the size around 45 kDa. We have confirmed that there is no premature stop codon
in pColdI-*pfa D*. We will perform experiments to confirm whether there is unexpected protease
activity or we may optimize the condon near the suspected premature ending site.

![](https://static.igem.wiki/teams/4131/wiki/result/result12.png)

![](https://static.igem.wiki/teams/4131/wiki/result/result13.png)

Figure 12: The Coomassie blue staining showed the induction of pfa D
proteins.

# Cloning pSTV28-*pfa B*-*pfa C*-*pfa E*

# Major experiment:

1. Amplifying the *pfa C* gene from the genome of *Moritella marina*.
2. Amplifying the *pfa B* (for DHA production), *pfa B'* (for EPA
   production) and *pfa E* genes from the biobricks ordered from IDT company.
3. Cloning *pfa B*, *pfa B'*, *pfa C* and *pfa E* genes into
   pSTV28 expression vector.
4. Expressing the pfa B, pfa B', pfa C and pfa E protein.

# Achievements:

Successfully amplifying the *pfa B* (for DHA production), *pfa B'* (for EPA production),
*pfa C* and *pfa E* genes, cloning *pfa E* into pSTV28 vector and expressing pfa E
protein.

# Associated result:

## Confirming the digested pSTV28 plasmids

  The pSTV28 plasmids were extracted from the cultured bacteria and subjected to EcoRI and
BamHI digestion. The gel electrophoresis result showed the sizes of restriction fragments of pSTV28 (4,
0.5, 0.5 kb).

![](https://static.igem.wiki/teams/4131/wiki/result/result14.png)Figure 13: The agarose image shows the digestion result of
pSTV28.

## Amplifying the *pfa B* by PCR

  To clone the *pfa B* (~2.6 kb) from the synthesized biobrick by the company, we
designed the forward primer with a restriction enzyme EcoRI site at the 5' terminal, while the 3'
terminal of reverse primer is ended by restriction enzyme BamHI site. The PCR amplification is performed
by the designed primer pair and pfu DNA polymerase.

  The primer sequence are listed below:

  EcoRI-*pfa B*-F: GAATTCATGACGGAATTAGCTGTTAT

  BamHI-*pfa B*-R: GGATCCTTATTTGTTCGTGTTTGC

  The gel electrophoresis result showed the size of *pfa B* amplicon (2.6 kb) as
expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result15.png)Figure 14: The agarose image shows the amplicon of *pfa B*.

## Amplifying the *pfa B'* by PCR

  To clone the *pfa B'* (~2.3 kb) from the gene block synthesized by the company, we designed
the forward primer with a restriction enzyme EcoRI site at the 5' terminal, while the 3' terminal of
reverse primer is ended by restriction enzyme BamHI site. The PCR amplification is performed using the
designed primer pair and pfu DNA polymerase.

  The primer sequences are listed below:

  EcoRI-*pfa B* EPA-F: GAATTCATGGAACAAACGCCTAA

  BamHI-*pfa B* EPA-R: GGATCCTTAGACTTCCCCTTGAAG

  The gel electrophoresis result showed the size of *pfa B'* amplicon (2.3 kb) as
expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result16.png)Figure 15: The agarose image shows the amplicon of *pfa B'*.

## Amplifying *pfa C* by PCR:

  To clone the *pfa C* (~6 kb) from the genome of *Moritella marina*, we designed
the forward primer with a ribosome binding site (RBS) and a restriction enzyme BamHI site at the 5'
terminal, while the 3' terminal of reverse primer is ended by restriction enzyme SacI site. The PCR
amplification was performed using the designed primer pair and pfu DNA polymerase.

  The primer sequences are listed below:

  BamHI-RBS-*pfa C*-F:

  GGATCCGGAGATATACCATGGAAAATATTGCAGTAGTAGG

  SacI-*pfa C*-R: GAGCTCTTACGCTTCAACAATACTTAAAAC

  The gel electrophoresis result showed the size of *pfa C* amplicon (6 kb) as expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result17.png)Figure 16: The agarose image shows the amplicon of *pfa C*.

## Amplifying *pfa E* by PCR

  To clone the *pfa E* (~0.8 kb) from the synthesized biobrick by company, we designed
the forward primer with a ribosome binding site (RBS) and a restriction enzyme SacI site at the 5'
terminal, while the 3' terminal of reverse primer is ended by restriction enzyme HindIII site. The PCR
amplification was performed using the designed primer pair and pfu DNA polymerase.

  The primer sequences are listed below:

  SacI-RBS-*pfa E*-F:

  GAGCTCGGAGATATACCATGTACAGCGGCGTAAAA

  HindIII-*pfa E*-R: AAGCTTCTATTTAGCGTCAGGTTTAAAAT

  The gel electrophoresis result showed the size of *pfa E* amplicon (0.8 kb) as
expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result18.png)Figure 17: The agarose image shows the amplicon of *pfa E*.

## The TA cloning of *pfa E* and sequencing confirmation

  After gel electrophoresis, the *pfa E* amplicon was purified from agarose gel and
ligated with TA-vector. The ligated TA-*pfa E* was then transformed into *E. coli* DH5α
strain, and the colonies after antibiotic (Amp) selection were subjected to colony PCR.

![](https://static.igem.wiki/teams/4131/wiki/result/result19.png)Figure 18: The agarose image shows the colony PCR result of
TA-*pfa E*.

  The clones showing expected amplicon size (0.8 kb) in colony PCR were then expanded for
TA-*pfa E* plasmid extraction, and the extracted TA-*pfa E* plasmids were subjected to
restriction enzyme digestion by HindIII.

![](https://static.igem.wiki/teams/4131/wiki/result/result20.png)Figure 19: The agarose image shows the digestion result of
TA-*pfa E*.

  The clones with correct restriction digestion map (2.7 kb for TA vector and 0.8 kb for
*pfa E*) were subjected to sequencing confirmation.

![](https://static.igem.wiki/teams/4131/wiki/result/result21.png)Figure 20: The electrogram shows the Sanger sequencing result of
TA-*pfa E*.

  Finally, we sequence-confirmed the TA-*pfa E*.

## The cloning of *pfa E* into pSTV28 vector

  The *pfa E* amplicon was excised from the TA-*pfa E* vector by SacI and HindIII
digestion, and the purified amplicon was ligated into pSTV28 vector. The colonies grown after standard
transformation were examined by colony PCR using a primer pair recognizing SacI-RBS-*pfa E*-F and
HindIII-*pfa E*-R. The size of the target amplicon of colony PCR is 0.8 kb in length.

  The primer sequences are listed below:

  SacI-RBS-*pfa E*-F:

  GAGCTCGGAGATATACCATGTACAGCGGCGTAAAA

  HindIII-*pfa E*-R: AAGCTTCTATTTAGCGTCAGGTTTAAAAT

![](https://static.igem.wiki/teams/4131/wiki/result/result22.png)Figure 21: The agarose image shows the colony PCR result of
pSTV28-*pfa E*.

  The pSTV28-*pfa E* vectors were then purified from colonies and confirmed by Sanger
sequencing.

![](https://static.igem.wiki/teams/4131/wiki/result/result23.png)Figure 22: The electrogram shows the Sanger sequencing result of
pSTV28-*pfa E*.

## The protein expression of pfa E

  We transformed pSTV28-*pfa E* into *E. coli* BL21 strain and induced protein
expression through culturing bacteria in TB medium with 1 mM IPTG at 37 °C for 4 hr. The bacteria was
then harvested and subjected to SDS-PAGE analysis and Coomassie blue staining. The predicted size of
pfa E protein is 32 kDa. The staining result clearly showed the induction of pfa E proteins.

![](https://static.igem.wiki/teams/4131/wiki/result/result24.png)Figure 23: The Coomassie blue staining showed the induction of
pfa E proteins.

  To optimize the protein expression, we performed a time course examination. The protein
expression was induced by culturing bacteria in TB with 1 mM IPTG at 37 °C for 2, 4 and 8 hr. All collected
samples were subjected to SDS-PAGE analysis and Coomassie blue staining.

![](https://static.igem.wiki/teams/4131/wiki/result/result25.png)

![](https://static.igem.wiki/teams/4131/wiki/result/result26.png)

Figure 24: The Coomassie blue staining showed the induction of pfa E
proteins.

  Because the induction was inefficient, we further optimized the protein expression by
extending the inducing time. The protein expression was induced by culturing bacteria in TB with 5 mM
IPTG at 37 °C for 8, 18 and 24 hr. All collected samples were subjected to SDS-PAGE analysis and
Coomassie blue staining. The staining result showed the induction of pfa E proteins around 32 kDa.

![](https://static.igem.wiki/teams/4131/wiki/result/result27.png)

![](https://static.igem.wiki/teams/4131/wiki/result/result28.png)

Figure 25: The Coomassie blue staining showed the induction of pfa E
proteins.

# Cloning pET28a-*AccBC*-*AccD1E*

# Major experiment:

1. Amplifying the *AccBC* and *AccD1E* genes from the biobrick synthesized by the company.
2. Cloning *AccBC* and *AccD1E* genes into pET28a expression vector.
3. Expressing ACCBC and ACCD1E protein.

# Achievements:

Successfully amplifying the *AccBC* and *AccD1E* genes, cloning *AccD1E*
into pET28a vector and expressing ACCD1 protein.

# Associated result:

## Confirming the digested pET28a plasmids

  The pET28a plasmids were extracted from the cultured bacteria and subject into SspI and SapI
digestion. The gel electrophoresis result showed the sizes of restriction fragments of pET28a (3.6, 1.2,
0.6 kb).

![](https://static.igem.wiki/teams/4131/wiki/result/result29.png)Figure 26: The agarose image shows the digestion result of
pET28a.

## Amplifying *AccBC* by PCR

  To clone the *AccBC* (~1.8 kb) from the gene block synthesized by the company, we
designed a forward primer with a ribosome binding site (RBS) and a restriction enzyme NdeI site at the
5' terminal, while the 3' terminal of reverse primer is ended by restriction enzyme EcoRI site. The PCR
amplification was performed using the designed primer pair and pfu DNA polymerase.

  The primer sequences are listed below:

*AccBC*-F: CATATGATGTCAGTCGAGACTAGGAA

*AccBC*-R: GAATTCTTACTTGATCTCCAGG

  The gel electrophoresis result showed the size of *AccBC* amplicon (1.8 kb) as
expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result30.png)Figure 27: The agarose image shows the amplicon of *AccBC*.

## The TA cloning of *AccBC* and sequencing confirmation

  After gel electrophoresis, the *AccBC* amplicon was purified from agarose gel and
ligated with TA-vector. The ligated TA-*AccBC* was then transformed into *E. coli* DH5α
strain, and the colonies after antibiotic (Amp) selection were subjected to colony PCR.

![](https://static.igem.wiki/teams/4131/wiki/result/result31.png)Figure 28: The agarose image shows the colony PCR result of
TA-*AccBC*.

  The clones showing expected amplicon size (1.8 kb) in colony PCR were then expanded for
TA-*AccBC* plasmid extraction, and the extracted TA-*AccBC* plasmids were subjected to
restriction enzyme digestion by NdeI and EcoRI.

![](https://static.igem.wiki/teams/4131/wiki/result/result32.png)Figure 29: The agarose image shows the digestion result of
TA-*AccBC*.

  The clones with correct restriction digestion map (2.7 kb for TA vector and 1.8 kb for
*AccBC* ) were subjected to sequencing confirmation.

![](https://static.igem.wiki/teams/4131/wiki/result/result33.png)Figure 30: The electrogram shows the Sanger sequencing result of
TA-*AccBC*.

  Finally, we sequence-confirmed the TA-*AccBC*.

## Amplifying *AccD1E* by PCR

  To clone the *AccD1E* (~2 kb) from the gene block synthesized by the company, we
designed a forward primer with a ribosome binding site (RBS) and a restriction enzyme EcoRI site at
the 5' terminal, while the 3' terminal of reverse primer is ended by restriction enzyme XhoI site. PCR amplification was performed using the designed primer pair and pfu DNA polymerase.

  The primer sequences are listed below:

*AccD1E*-F: GAATTCGGAGATATACCATGACCATTT

*AccD1E*-R: CTCGAGCTAGAAGAAATTCACATTC

  We then performed PCR to amplify the target fragment. The result showed that the amplicon
sizes of *AccD1E* (2 kb) as expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result34.png)Figure 31: The agarose image shows the amplicon of *AccD1E*.

## The TA cloning of *AccD1E* and sequencing confirmation

  After gel electrophoresis, the *AccD1E* amplicon was purified from agarose gel and
ligated with TA-vector. The ligated TA-*AccD1E* was then transformed into *E. coli* DH5α
strain, and the colonies after antibiotic (Amp) selection were subjected to colony PCR.

![](https://static.igem.wiki/teams/4131/wiki/result/result35.png)Figure 32: The agarose image shows the colony PCR result of
TA-*AccD1E*.

  The clones showing expected amplicon size (2 kb) in colony PCR were then expanded for
TA-*AccD1E* plasmid extraction, and the extracted TA-*AccD1E* plasmids were subjected to
restriction enzyme digestion by EcoRI and Xhol.

![](https://static.igem.wiki/teams/4131/wiki/result/result36.png)Figure 33: The agarose image shows the digestion result of
TA-*AccD1E*.

  The clones with correct restriction digestion map (2.7 kb for TA vector and 2 kb for
*AccD1E*) were subjected to sequencing confirmation.

![](https://static.igem.wiki/teams/4131/wiki/result/result37.png)Figure 34: The electrogram shows the Sanger sequencing result of
TA-*AccD1E*.

  Finally, we sequence-confirmed the TA-*AccD1E*.

## The cloning of *AccD1E* into pET28a vector.

  The *AccD1E* amplicon was excised from the TA-*AccD1E* vector by EcoRI and XhoI
digestion, and the purified amplicon was ligated into pET28a vector. The colonies grown after standard
transformation were examined by colony PCR using a primer pair recognizing *AccD1E*-1721 F and T7
terminator. The size of the target amplicon of colony PCR is 0.3 kb in length.

  The primer sequences are listed below:

*AccD1E* 1721-F: ATCGTATCTGGCAACCCAAC

  T7-terminator: GCTAGTTATTGCTCAGCGG

![](https://static.igem.wiki/teams/4131/wiki/result/result38.png)Figure 35: The agarose image shows the colony PCR result of
pET28a-*AccD1E*.

  The pET28a-*AccD1E* vectors were then purified from colonies and confirmed by Sanger
sequencing.

![](https://static.igem.wiki/teams/4131/wiki/result/result39.png)Figure 36: The electrogram shows the Sanger sequencing result of
pET28a-*AccD1E*.

## The protein expression of ACCD1 and ACCE

  We transformed pET28a-*AccD1E* into *E. coli* BL21 strain and induced protein
expression through culturing bacteria in TB medium with 0.4 mM IPTG at 37 °C for 2, 3 and 4 hr. The
bacteria was then harvested and subjected to SDS-PAGE analysis and Coomassie blue staining. The
predicted size of ACCD1 and ACCE protein are 60 and 10 kDa, respectively. The staining result clearly
showed the induction of ACCD1 protein around 60 kDa. The ACCE protein expression will be further
examined by using 15% gel in SDS-PAGE analysis and Coomassie blue staining.

![](https://static.igem.wiki/teams/4131/wiki/result/result40.png)Figure 37: The Coomassie blue staining showed the induction of
ACCD1 protein.

# Triple antibiotic selection

# Major experiment:

1. Conducting double antibiotics selection to confirm the ideal concentration of
   antibiotics for pColdI and pSTV28 vectors.
2. Conducting triple antibiotics selection to confirm the ideal concentration of
   antibiotics for pColdI, pSTV28 and pET28a vectors.

# Achievements:

Successfully determine the ideal concentration of antibiotics for pColdI, pSTV28 and
pET28a vectors to co-exist in *E. coli.*

# Associated result:

## Conducting double antibiotics selection

  To confirm that the pColdI (Ampicillin resistant) and pSTV28 (Chloramphenicol resistant) can
be co-expressed in the bacteria to establish the pfa megasynthase expression system, we first performed
double antibiotic selection for pColdI and pSTV28 vectors.

  We started by fixing the concentration of ampicillin at 50 μg/ml, which is generally applied
to select plasmids with Amp-resistance. The transformed copy number of plasmid was fixed at
109. A range of chloramphenicol concentrations from 5 to 20 μg/ml was selected according to
the reports using the pSTV28 vector as expression vector.

  The selection result showed an apposite number of colonies at 10 μg/ml of
chloramphenicol. Therefore, we selected the Amp (50 μg/ml) and Cm (10 μg/ml) for the double antibiotic
selection.

| Copy number of each plasmid: 109 ; Ampicillin: 50 μg/ml | | | | | |
| --- | --- | --- | --- | --- | --- |
| Chloramphenicol | Colony number | | | Mean | SD |
| 5 μg/ml | 232\* | 1240 | 1244 | 1242 | 2082 |
| 10 μg/ml | 255 | 360 | 542 | 386 | 145 |
| 20 μg/ml | 206 | 437 | 937\* | 322 | 163 |

\*outliers excluded from the average.

  To further confirm that the two kinds of plasmids are co-expressed in the colonies, we
randomly selected some of the colonies and purified the plasmids from these colonies. The purified
plasmids were digested with restriction enzyme SapI. The gel electrophoresis result showed the sizes of
restriction fragments of pColdI (5.7 kb) and pSTV28 (4.2, 0.9 kb) as expected.

![](https://static.igem.wiki/teams/4131/wiki/result/result41.png)Figure 38: The agarose image shows the digestion result of pColdI
and pSTV28.

## Conducting triple antibiotics selection

  To confirm that the pfa-expressing vectors (pColdI and pSTV28) can be co-expressed with
pET28a (kanamycin resistant) vector, we performed triple antibiotic selection.

  According to the result of double antibiotics selection, we started the triple antibiotic
selection by fixing the concentration of Amp (50 μg/ml) and Cm (10 μg/ml). However, this condition was
too harsh when adding kanamycin as the third antibiotic, and only a few colonies survived. We then
decreased the concentration of chloramphenicol to 5 μg/ml, and performed triple antibiotics selection
with 1.25 μg/ml, 2.5 μg/ml and 5 μg/ml kanamycin.

  The result showed an apposite number of colonies at 5 μg/ml of kanamycin. Therefore,
we selected Amp (50 μg/ml), Cm (5 μg/ml) and Kan (5 μg/ml) for the triple antibiotic selection.

| Copy number: 109 ; Ampicillin: 50 μg/ml, Chloramphenicol: 5 μg/ml | | | | | |
| --- | --- | --- | --- | --- | --- |
| kanamycin | Colony number | | | Mean | SD |
| 1.25 μg/ml | > 1000 | > 1000 | > 1000 | X | X |
| 2.5 μg/ml | 134 | 252 | 534 | 193 | 83 |
| 5 μg/ml | 79 | 139 | 179 | 132 | 50 |

  To further confirm that the three kinds of plasmids are co-expressed in the colonies,
we randomly selected some colonies and purified the plasmids from these colonies. The purified
plasmids were digested with restriction enzymes XbaI and BamHI. The gel electrophoresis result showed
the sizes of restriction fragments of pColdI (4.4, 1.2 kb), pSTV28 (3.2, 1.4, 0.5 kb) and pET28a (5.6
kb) as expected.
The five colonies have been conducted plasmid extraction and digestion, and the results are shown in below.

![](https://static.igem.wiki/teams/4131/wiki/result/result42.png)Figure 39: The agarose image shows the digestion result of
pColdI, pSTV28 and pET28a.

  Through the double and triple antibiotic selection, we set up conditions which allow pColdI
and pSTV28, or pColdI, pSTV28 and pET28a vectors to co-exist in *E. coli*. Therefore, we can
co-transformed our engineering plasmids into *E. coli* and co-expressed the pfa megasynthase with ACC
enzymes to produce EPA.

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