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Background Information

The advent of modern medicine has also created a growing need for blood transfusions. Many modern treatments for various diseases are only possible due to advanced transfusion medicine. Especially emergency medical aid is heavily dependent on a constant supply of donor blood in order to save lives.

There are many aspects of blood that need to be considered before a patient can receive a transfusion since erythrocytes (red blood cells) show various antigens on their surface that could be recognized by the receiver’s immune system and thereby cause complications. The ABO antigens are the most widely known antigens of erythrocytes and together with the Rhesus factor are the most relevant antigens for transfusions. When only considering the ABO system and Rhesus factor there are the blood types A, B, AB and O, either with or without the Rhesus factor antigen (Rhesus factor positive or negative). Generally, an individual always has antibodies against antigens that are not expressed on the surface of the own erythrocytes.

The ABO antigens consist of glycosilated proteins on the erythrocyte’s surface. Individuals with blood types A or B both have a distinct glycosilation that serves as an antigen. Individuals with blood type AB have a mix of proteins: some are glycosilated analogous to blood type A and some are glycosilated analogous to blood type B. The blood type O antigen (“H antigen”) is a primitive glycosilation that serves as a basis for the glycosilations that then form type A or B. It is important to note here that there are no antibodies against the H antigen and the anti-A and anti-B antibodies do not interact with this very basic glycosilation. Similarly, Rhesus factor antibodies do not interact with the Rhesus factor negative antigen. Thus, it is possible to transfer type O Rh- to individuals with any other blood group – type O Rh- is the universal donor.

Often, the blood type of patients needing emergency transfusions is unknown and tests would take too long or are not possible in the given situation. Thus, patients of unknown blood type receive the universal donor blood type O Rh-. This, however, creates a huge demand for blood of type O and clinics usually have to plan ahead carefully in order to always keep a sufficient stock of type O Rh- blood. Especially in crisis regions the stockpiling of type O Rh- blood is a difficult task.

Our Project

As we see it, there are three main ways to increase the amount of type O Rh- blood available to hospitals: (a) more individuals need to donate blood, (b) artificial blood is approved for use on humans, and (c) blood is converted to universal donor type.

Increasing the number of blood donors is more of a societal approach to a solution of this problem and indeed might have some advantages apart from only increasing the amount of donor blood. However, we think it is unlikely that more people will suddenly start donating blood since there have been many media campaigns encouraging the donation of blood in the past which did not solve the problem of shortages of universal donor blood. Also, the universal donor blood type O Rh- has only a low prevalence in the human population. Thus, it is not likely that the supply of universal donor blood would significantly increase if more people were donating blood.

The development of artificial blood is a very promising if not even the best solution to blood shortages. However, artificial blood is not available yet and clinical trials are only about to start in the coming years. Thus, artificial blood is a very good solution in the long run but will not save lives in the short term.

We think that the conversion of blood to type O Rh- is a very good solution until artificial blood becomes widely available. Blood conversion would not only solve the problem of shortages but also would increase the cost effectiveness of blood donations since blood of types other than O Rh- could be converted and used in emergency medical aid before it expires. Thus, we decided to focus our this year’s iGEM project on the development of an efficient and fast biological system for the conversion of blood.

Our project is mainly based on the works of Liu et al. (2007) and Shaikh et al. (2009) who have described enzymes (glucosidases) which are capable of the conversion of A and B antigens to O and a type similar to Oh, respectively. Thus, we focus on the conversion of Rh- blood in order to obtain type O Rh-.

The glycosilations that characterize each blood type can be enzymatically cleaved, partially or completely, by the glucosidases described in the aforementioned publications which results in a glycosilation that corresponds to the O or Oh antigen. We plan on using the following enzymes for our system (see also the illustration below):

• N-Acetyl-Galactosaminidase from Elisabethkingia meningosepticum - converts A to O
• α-Galactosidase from Bacteroides fragilis - converts B to O
• Endo-β-galactosidase from Clostridium perfringens - converts A and B to Oh

In order to obtain these enzymes for their utilization in our system, the corresponding genes will be overexpressed in Escherichia coli. We plan to tag the enzymes with protein-tags like His-tag in order to simplify the following purification process. Then the enzymes will be immobilized on a carrier matrix using fusion protein tags like Spy-tag or SNAP-tag. The assembled system will then be ready for the conversion of erythrocytes – which we plan to actually demonstrate over the course of the summer.

We also plan to expand the BioBrick Registry by developing BioBricks relevant for protein expression (e.g. promoters).

Graphical abstract of our project for iGEM 2014. With the help of our blood-type conversion system the A and B antigens of erythrocytes will be cleaved by up to three different glycosidases which will result in O or Oh antigens. Thus, we will create a method for the conversion of blood to type O which then can be used in emergency transfusions with receivers of unknown blood type.

Team

Team Tuebingen consists of about 20 motivated students of biology and biochemistry. We are supported by University of Tuebingen’s administration and faculty but are still self-organized and self-financed. Since our first participation in iGEM in 2012, we are supported by several professors working in disciplines related to synthetic biology who provide us with modern lab equipment and helpful advice.

Contact

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Sponsors

We want to warmly thank the following sponsors of our iGEM2014 project (in no particular order):