INTRODUCTION RESULT

HUMAN PRACTICE

KOREA U SEOUL

Kyengwoo Jang*, Jaywook Han, Minhwan Yu, Jihoon Jeong, Minseob Yoo, Junhong Jang, Hyeyeong Choi, Jihee Park, Juyoung Han, Hyunsong Kim, Jonghee Chun

Instructor : In-geol Choi, Dongha Kim / Advisor : Areum Goh (*Corresponding author e-mail : unjgs@korea.ac.kr)

PARTS DESIGN

CONCLUSION

REFERENCE 1. Hiroshi Miyamoto , Aizo Matsushiro et al. 1996. A carbonic anhydrase from the nacreous layer in oyster pearls. Proc Natl Acad Sci. p9657-9660. 2. Hiroshi Miyamoto et al. 2005 The Carbonic Anhydrase Domain Protein Nacrein is Expressed in the Epithelial Cells of the Mantle and Acts as a Negative Regulator in Calcification in the Mollusc Pinctada fucata. Zoological science p311-316. 3. Hyeok-Jin Ko, In-Geol Choi et al. 2012. Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes in Escherichia coli with a Novel Ligation Independent Cloning Vector Based on the Autotransporter YfaL. AEM. 10.1128

METHOD

Our team member, Hye young Choi, worked medical volunteering in the

Republic of Cote d'Ivoire. She visited the University of Bouaké as well, and

announced Life Science with iGEM competition: introduce synthetic

biology, the usage of registered bio-bricks, applications and features.

Korea_U_Seoul team decided to form an academic society of synthetic biology,

Korea University Association of Synthetic Biology (KUAS) ,for the purpose of sharing information,

researching, and spreading knowledge about synthetic biology to the public.

Sponsored by the Seoul Metropolitan Government, our team participated in the Seoul 2013 Nolto EXPO (amazing Saturday in Seoul EXPO) in June 22, 2013. We made posters for pubilcizing iGEM and explaining Synthetic biology to children.

Creative Challenger Program(CCP) is a Korea University's program which

encourages undergraduates to organize a team and research about their

concerned field. Korea_U_Seoul has been participating in CCP since 2010, sharing knowledge about iGEM and

synthetic biology for years

Domain

Cloning

We cloned 4 nacrein genes into pB4 vector, modified pET vector for protein purification. This vector had MBP protein tag for increasing the solubility of protein. Also, we cloned the genes into pATLIC for displaying nacrein protein in the cell face of E. coli.

Expression test

Cells of E. coli carrying nacrein genes in pB4 vector were cultivated in auto-inducing medium ZYM-5052 with ampicillin (100 μg/ml) for 7 h at 37°C or for 24 h at 16°C. The cells were harvested by centrifugation (13,500 x g for 1 min) and analyzed with SDS-polyacrylamide gel.

Protein purification

Cells were harvested and disrupted by sonication. After centrifugation, crude extract was loaded on HP column to purify proteins with Ni2+ affinity chromatography in LP system.

CaCO3 Precipitation Assay

10 mM NaHCO3, 10 mM CaCl2 and MBP-nacrein fusion proteins were prepared at time 0. We measured pH every 30 seconds at 25°C.

Carbonic Anhydrase Assay

The purified protein was dialyzed against 20 mM Tris-Cl (pH 8.0) buffer containing 1 mM ZnSO4. 12 ml of 20 mM Tris-Cl (pH 8.0) buffer, 0.2 mg enzyme and 8 ml of CO2-saturated water was mixed and pH was monitored at 0°C.

Figure 1: A schematic representation of nacrein domain

Pearl-coli: Escherichia coli converting CO2 into a pearl powder

Expression Test

Activity Test

The mechanism in which nacrein converts CO2 into HCO3- is the following:

CO2 + H2O → HCO3- + H+

If nacrein was active, pH should be dropped abruptly in short time by hydronium ion. But we found that pH decreased slowly. The slow decrease was resulted from natural decomposition of HCO3-. Repeat region of nacrein binds with Ca2+ ion. It means that Ca2+ cannot bind with CO3

2- ion to form CaCO3. If repeat region of nacrein had activity, pH would decrease slowly by natural decomposition of HCO3

-

Ca2+ + HCO3- → CaCO3 + H+

However, we found steep decrease, because of fast decomposition of HCO3-, which indicates that Ca2+ bound with CO3

2-. In short, repeat region of nacrein didn’t have activity.

Figure 3. SDS-PAGE of MBP (44 kDa)-fusion nacrein proteins. Lane 1, Protein size marker; lane 2, supernatant (S) of Nacrein WT (50 kDa); lane 3, Pellet (P) of Nacrein WT; lane 4, deleted repeat (40 kDa, S); lane 5, deleted repeat (P); lane 6, carbonic anhydrase (25 kDa, S); lane 7, carbonic anhydrase (P); lane 8, repeat domain (9 kDa, S); lane 9, repeat domain (P).

Figure 4. CA activity assay by pH change. NA: nacrein; CA: carbonic anhydrase; Tris: control

Figure 5. CaCO3 precipitation assay NA: nacrein; R: repeat domain; Tris: control

• We successfully expressed and purifed four mutants of nacrein. • We meausred CA activity of nacrein but it didn’t have activity. • We tried to check role of nacrein in precipitation of CaCO3 but it didn’t work.

Figure 2. iGEM part. Green curved arrow: pBAD promoter; circle: ribosome binding site (RBS); purple arrow: nacrein mutants with YfaL domain. YfaL domain was used to express nacrein on the cell surface of E. coli.

GLOBAL WARMING

Global warming is one of the most serious problem nowadays and caused by high levels of carbon dioxide and other gases in the atmosphere. To solve this problem, we need to decrease the level of CO2

NACREIN

Nacrein is enzyme from pearl oyster (Pinctada fucata). It has carbonic anhydrase (CA) activity which converts H2O and CO2 into HCO3

- and H+. It is proposed that nacrein contributes to nacreous layer formation.

Nacrein contains 2 domain, Repeat and CA. Repeat domain is binding site for Ca2+. We tried to display 4 parts on the cell surface of Escherichia coli acting as whole-cell biocatalysis.

pBAD BBa_K114600 YfaL_Nacrein(WT)

pBAD BBa_K114601

YfaL_Nacrein CA

pBAD BBa_K114602 YfaL_Nacrein DR

pBAD BBa_K114604

YfaL_Nacrein R

We confirmed that four mutant proteins were expressed in soluble part. After confirmation, we purified the proteins.