Intrinsic Factor-y An alternative treatment for Persinicous Anemia

Meet Our TEAM!

Introduction

Pernicious Anemia, which is recognized as an autoimmune

disorder, is caused by atrophic damage of the gastric body mucosa in

which results loss of parietal cells that express a 60-kd glycoprotein

called intrinsic factor (IF) (1,2). Function of IF is binding and transporting

vitamin B12 in small intestine (3). Main Cause Vitamin B12 deficiency. • Cannot be synthesized in the human body and it must be taken by

diet.

• The minimum daily requirement of vitamin B12 is about 2.5 µg. (3) Absorption of B12

Abstract Pernicious anemia is a type of megaloblastic anemia occurs due

to lack of Intrinsic Factor, which is a protein responsible for the

absorption of vitamin B12. Our aim as ITU MOBGAM IGEM Team, is to

design a bacterium that is capable of surviving in small intestine and

secreting Intrinsic Factor dependent on pH. Also, we design a genetic

circuit for controlling the overgrowth and containment of bacteria.

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Human Practice As we are ITU_MOBGAM_Turkey team, our one of the goals was publicizing iGEM through our university, Istanbul and Turkey. For this, • We use social networks; Twitter, Facebook, and our-non iGEM

website • We interview with people with a questions: What is iGEM? and What

is Synthetic Biology? • We pressed articles in our university newspaper and journal called

Science & Future • We designed a poster and hanged in our university

Besides those, we appealed professionals’ and patients’ opinions about our project. • We have interviewed with Dr. Aslıgül Kendirci (Drug Development

Director of Roche and also PDGA Country Head Turkey, Middle East and North Africa)

In order to meet other iGEM teams in Turkey, we attended first National iGEM Teams Meeting in Ankara, and we hosted second National iGEM Teams Meeting in Istanbul

Module 1: Secretion of GIF GIF protein fused with OmpA signal peptide was synthesized. Secretion was planned to validate with Western Blot analysis from culture supernatant.

Module 2: Purification and Characterization of GIF Recombinant GIF protein was planned to clon into GST-tag vector for purification. Then, fuctionality of recombinant protein was planned to analyze in meaning of capability of binding B12

Source of Vitamin B12 Stomach

(Digestion) Revealing Vitamin B12 content

Binding with R-protein (haptocorrin)

(R-protein/B12 complex)

Duodenum

Transfer of B12 from R-protein to intrinsic factor

(Intrinsic factor/B12 complex)

Ileum

Recognition of IF/B12 by receptor on epithelial cells

Absorption of IF/B12 complex by epithelial cells

Treament Patients with pernicious anemia require a life-long treatment. • Obtain B12 by injection or pill

Symptoms General symptoms • Weakness • Abnormal fatique • Sore tongue

Effects on mainly three system • Blood • Gastro-intestinal

tract Gastric ulcer • Nervous system Abdorminal pains

Modelling As you can see in the diagram given below, MazE protein expression is stimulated by glucose, MazF protein expression is always presented under weak constitutive promoter; however, it can be stimulated by AI-2. We assign glucose and cell density as parameters and want to find the value of those where MazE/MazF is greater than 1. Our cell can only be alive in such region.

Team Members: Recep Erdem Ahan, Behide Saltepe, Eren Sahin, Tugce Onur, Busra Ahata, Bogac Ercig, Cahit Haktan

Caglar, Can Gurkaslar, Deniz Kinik, Guleycan Lutfullahoglu, Guliz Otkiran, Gulsima Dilek Usluer Mahir Bozan,

Mehmet Akdag, Nazif Tasbas, Seda Karabacak, Sibel Karaman

Advisors: Dr. Oyku Irigul Sonmez, Dr. Deniz Sahin

Instructors: Assoc. Prof. Dr. Ayten Yazgan Karatas, Assoc. Prof . Dr. Fatma Nese Kok

E-mail: igem.itu@gmail.com

Our non-iGEM website: igem.itu.edu.tr

Address: MOBGAM, İstanbul Teknik Üniversitesi, Ayazağa Kampüsü, 34469 Maslak-İstanbul

Module 3: pH Dependent expression of GIF GIF fused with OmpA signal peptide was planned clon upstream of nhaA promoter. It would provide expression of GIF only in small intestine. Expression of was planned to validate with Western Blot analysis. Culture would be grown basic pH.

Module 4: Kill switch Growth of bacterium was planned to restricted with two parameters: glucose for growth only in small intestine, and AI-2 for growth with a limited number. MazE-MazF module was planned established in bacterium.

References 1. Vanella, L. et al. (2012). Systematic review:

gastric cancer incidence in pernicious anaemia.

AP&T (37), 375-382.

2. Kaplan, S. & O’Connor, S. (n.d.) Final

diagnosis-anemia. Retrieved from 1 August

2013 from

http://path.upmc.edu/cases/case428/dx.html

3. Sowers, Helen M. (n.d.) Megaloblastic

Anemia. s.l. : California Association for

Medical Laboratory Technology. DL-975.

Figure 2: The part

Figure 3: Agarose gel results of parts double cut ( Lane right to left: Empty, OmpA-GIF+B0015, OmpA-GIF, B0034, Marker)

Figure 4: Agarose gel result of BamHI and XhoI double cut of plasmid isolated from colonies which are transformed with ligation product

Figure 7: PCR amplification of nhaA promoter at 59 0C annealing temparature

Figure 6: Agarose gel results of amplification nhaA promoter from DH5α strain via gradient PCR

Figure 5: The part Figure 8: The part

Figure 10: Validation of ligation reaction Figure 9: EcoRI cut of first ligation reaction products

Figure 1: Absroption Mechanism of B12

Figure 11: Logical scheme of process Figure 12: Equations