BRAWIJAYA UNIVERSITY

We used E. coli strain DH5a for plasmid replication and BL21 for high level expression of our protein of interest. We would like to build a non invasive home diagnostic kit for Dengue Fever. Engineered bacteria will produce scFv antibody which has ability against glycoprotein of DEN virus envelope which contained in urine as sample. Our part contains osmY coding reading region, histidine, TEV cleave site, and scFv antibody. osmY coding region will produce peptide as signal which work due to osmotic shock. Histidine as protein tag for the nickel column purification. TEV cleave site will cut the scFV antibody from other protein. scFv which contained in our part consist of light chain, heavy chain, and Myc tag. We work at laboratory which has safety level 2 procedures. We wear rubber hand gloves, google glasses, and safety jacket. We sterilize all the waste based on the safety procedures that we used then wash our hands after work. We will put our antibody inside a small enclosed device that ordinary people can buy, like a test pack for pregnancy. It will has a very low risk. It will not harm human health, because it will detects the envelope glycoprotein of Dengue Virus that present in urine. If it reveals any threats in the future, we will withdraw all the products from market and examine what is wrong with our product or its system that we used then we fix it. From the device, it will give a result form the test. If it is positive, we also build a software to record personal data from patient which will be saved in the central database of the Ministry of Health.

Our Wet Lab

There are several step that we made to create

1. Design

We design envelope glicoprotein DENV 1 and DENV 2 antigen also there EDE1C8 antibody include TEV protease. That design is sequence of nucleotide that sticky-ended.

2. CoCulture

Escherichia coli strain DH5α is not a pathogen, and was developed for laboratory cloning use. This strain was developed by D. Hanahan as a cloning strain with multiple mutations that enable high-efficiency transformations. The genomic structure of this strain is a singular circular chromosome consisting of 4,686,137 nucleotides, 4359 genes, and 4128 protein encoding genes. This strain also contains plasmids, and has the ability to accept plasmid insertion exceptionally well. Escherichia coli is the most commonly used host for high-yield expression of recombinant protein, usually by exploiting the high promoter specificity and transcriptional activity of bacteriophage T7 RNA polymerase. However, several Escherichia coli host proteins also contain nonconsecutive histidine residues that exposed to the surface of their ternary structure (Robicon, et al, 2011). Escherichia coli strain BL21 and strain DH5α have cultured in Luria Bertani Agar media for 24 hours at 37°C. Then, we inoculate them in Luria Bertani Broth for 24 hours at 37°C. All media were supplemented with appropriate antibiotics as follows: chloramphenicol 170 μg/ml for maintaining the mini F′ plasmid pSB1C3 10 μg/ml for maintaining EDE1C8 constructs. Luria Bertani (LB) is a widely used bacterial culture medium but it has its origins in the field of bacteriophage genetics. LB medium is a rich medium that is commonly used to culture members of the Enterobacteriaceae as well as for coliphage plaque assays. LB and related media (such as SOC) are used extensively in recombinant DNA work and other molecular biology procedures. Often an antibiotic is added to the sterilized medium to select for cells that contain a specific genetic element such as a plasmid, a transposon, or a gene disruption via an antibiotic resistance cassette (MacWilliams and Liao, 2013).

3. Competent Cell

We made a competent cell with two different technique that published by Cohen, et al (1972) and CCBM 80 technique from iGEM. The first technique is frequently used to prepare batches of competent bacteria that yield 5x106 to 2x107 transformed colony/µg of supercoiled plasmid DNA. Also, we did not preserved at -70°C because we bewared that competent bacteria through some deterioration in the efficiency of transformation during prolonged storage. For efficient transformation, the essential number of viable cells not exceed 108 cells/ml that equivalent to an OD600 ~0.4. The culture The second technique used CCMB 80 for

4. Cloning

To make plain the progress of our cloning, the results will not be presented chronologically, but rather in a logical procedure, starting with the envelope glycoprotein DENV 1 and DENV 2 antigen also there EDE1C8 antibody include TEV protease constructs . This is not an specific list, but exclusively a presentation to make plain our work and give insight into our thoughts. The antigen parts of DENV 1, DENV 2, from E. coli was already present in parts registry in following are K_1823004 and K_182_3005. The antibodies part of EDE1c8TEV, EDE1C8, and EDE1C8(with promotor) in following are K_1823001, K_1823002, and , K_1823003. The 2014 Waterloo iGEM Team attemped the pSB1C3 backbone has been made more versatile by turning it into a shuttle vector. By cloning a Staphylococcal selective marker (erythromycin resistance gene) and origin of replication, this part has been improved to be used in both E. coli and Staphylococcal organisms. The Staphylococcal parts have been incorporated into the backbone between the VR and the pMB1 replication origin in pSB1C3.

5. Miniprep

The minipreparation of plasmid DNA, or miniprep, is a small-scale isolation of plasmid DNA from bacteria.

6. Restriction

For the first step of 3A assembly, we made some restriction step of DNA plasmid molecule using EcoR1 and Pst1restrction enzyme. This step a really important in gene construction. According to Wilson (2005) DNA molecule from current organism will give the same set of fragments when digested with a particular enzyme so it is possible to reproducibly divide its genome into a large number of small fragment, each approximately the size of a single gene. we used EcoR1 restriction enzyme, which recognises the 5-GAATTC-3, will produce fragments each of which is on average just over 4 kb. This makes subsequent steps more manageable, since a smaller number of those fragments need to be cloned and subsequently analysed.

7. Ligation

DNA plasmid from restriction step will paired each other in DNA Plasmid Backbones from iem. According to Wilson (2005) The DNA products resulting from restriction digestion to form sticky ends may be joined to any other DNAfragments treated with the same restriction enzyme. Thus, when the two sets of fragments are mixed, base-pairing between sticky ends will result in the annealing together of fragments that were derived from different starting DNA. so these pairing are transient, owing by the weakness of hydrogen bonding between the few bases in the sticky end. to stabilised these fragment we using an ligation enzyme called T4 DNA ligase since ligation reconstructs the site of cleavage, recombinant molecules produced. By ligation of sticky ends can be cleaved again at the ‘joins’, using the same restriction enzyme that was used to generate the fragments initially.

8. 3A Assembly

We made assembly of DNA plasmid, we rearrange the sequences of DNA plasmid and assembling two BioBrick parts together.

9. Transformation

For early step of DNA manipulation, we insert the plasmid of competent cell that we already made before, the inserting process of current plasmid to the bacteria called transformation. we using plasmid backbones pSB1C3 and put in into Escherichia coli strain DH5α. The plasmid is much smaller than a natural plasmid, which makes it more resistant to damage by shearing, and increases the efficiency of uptake by bacteria (Wilson,2005)

REFERENCE

Robichon, C., J. Luo, T. B. Causey, J. S. Benner, J. C. Samuelson. 2011. Engineering Escherichia coli BL21(DE3)

Derivative Strains To Minimize E. coliProtein Contamination after Purification by Immobilized Metal Affinity Chromatography. 77(13): 4634-4646.

MacWilliams, M. P. and M. K. Liao. 2013. Luria Broth (LB) and Luria Agar (LA) Media and Their Uses Protocol. ASM Microlibrary.

Wilson, keith . john walker .2005. biochemistry and molecular biology. Cambriedge university press. USA