Gastric Intrinsic Factor

Gastric intrinsic factor (GIF), trans-Cbl II, haptocorrin are protein that function to uptake, transport and store the vitamin B12. gif gene is located on chromosome 22. Gastric intrinsic factor consists of 399 amino acids residues in addition %15 carbohydrates content. Its molecular mass is 60-kDa according to gel filtration. (7)

Type II secretion System

Gram-negative bacteria have six types of secretion systems: type I, and type II secrete the protein in the extracellular space. Type I secretion system secretes the protein in a single-step whereas Type II secretion system uses two-step secretion. We use type II secretion system in our project. There are three different pathways in Type II secretion system: twin-arginine translocation (TAT), SecB-dependent and signal recognition particle (SRP). Different signal sequences are recognized by those pathways and helps to secretion of proteins through the periplasmic space of bacterium. Signal sequences are cleaved after the secretion. Proteins are folded while secreted extracellularly. Secretion from periplasmic space to extracellular space is unknown; however, different strategies were developed in laboratory such as co-expression of kil gene. (8)

7.Mathews, F. S. et al. (2007). Crystal structure of human intrinsic factor: cobalamin complex at 2.6 Å resolution. Proc. Natl. Acad. Sci. USA 104 (44), 17311-17316.

8.Mergulhao, F. J. M., Summers, D. K. & Monteiro, G. A. (2005). Recombinant protein secretion in E. coli. Biotechnology Advances (23), 177-202.

Method

In the first stage of the project, the production of intrinsic factor protein is aimed. For the production of IF, which is a protein composed of 399 amino acids encoded by 1254 nucleotides, gif gene will be reconstructed according to the iGEM RFC [10] standard with OmpA secretion signal on N-terminus and synthesized by Integrated DNA Technology Company as it will be contained in a cloning vector.

In the second stage of the project, recombinant IF protein is aimed to be secreted into the extracellular matrix of E. coli cells. With the help of 3A assembly strategy, gif gene transferred into a new vector system which contains a T7 promoter, strong RBS, and transcription terminator. ImmunoBlot will be used in order to examine efficiency of secretion.

Result

In order to examination of secretion of GIF protein with OmpA signal, a composite part which contains T7 promoter, RBS (B0034), GIF gene with OmpA signal sequence(OmpA-GIF) and double terminator (B0015) has been planned to ligate. First step, GIF-OmpA and doubled terminator (OmpA-GIF + B0015) were ligated (Figure 1). Second step, OmpA-GIF + B0015 planned ligated into B0034 part. Therefore, B0034 was cut with S and P; also, OmpA-GIF + B0015 was cut with X and P. Unexpected 300 bp DNA fragment was observed in B0034 double cut. Bands that observed in lane loaded with DNA from OmpA-GIF + B0015 double cut were expected (Figure 1).

Figure 1: Agarose gel results of parts double cut ( Lane right to left: Empty, OmpA-GIF+B0015, OmpA-GIF, B0034, Marker)

Introduction

Purification of a protein is required prior to investigation of its structure and action mechanism. However, it is not an easy task to separate single protein from other 10,000 different proteins (approximately) in the cell. To overcome this, there are different methods and strategies were developed.(1)

Usage of recombinant fusion proteins is a widely used strategy for purification of proteins. In this strategy, a tag with a known size is used as the tag for the protein of interest and allows its efficient purification and detection by affinity chromatography. Two commonly used tags are glutathione-S-transferase (GST) tag and 6 x Histidine residues (His)6 tag. (2)

GST tag is a protein that is composed of 211 amino acids and has a molecular weight of 26 kDa. DNA sequence that encodes GST is transferred into an expression vector in order to produce recombinant fusion proteins. GST tagged fusion proteins involve a GST protein attached to their N-terminus. GST tag provides higher expression rates and solubility to the recombinant protein that is fused. Moreover, detection of the protein is possible due to the substrate of GST, glutathione (GTH). (3)

Methods

Expression and characterization of Intrinsic Factor (IF) protein was aimed in this module of the project. For this purpose, Gastric intrinsic factor (gif) gene that encodes for intrinsic factor (IF) protein was chosen.

First of all, polymerase chain reaction (PCR) is going to be performed for both amplification of gif gene and addition of restriction enzyme digestion sites (BamHI and XhoI). Then, PGEX-6P1 expression vector will be digested by BamHI and XhoI restriction enzymes and the resulting sticky ends of the vector will be ligated with the sticky ends of gif gene.

Second, after the completion of insertion of gif gene into the vector, expression vector will be transformed into the E. coli DH5alfa strain. Then, bacterial growth will be achieved in order to express GST-tagged intrinsic factor protein.

Third, intracellular IF protein will be isolated and expression of the intrinsic factor protein will be confirmed by SDS-PAGE. Following with the confirmation that IF is successfully expressed, IF protein will be isolated by using affinity chromatography. In this process, GST-tagged IF protein will be purified with the help of its affinity to bind to GSH containing supporting matrix. After the purification of GST-tagged If protein, its purity will be confirmed by applying another SDS-PAGE. If desired purity was achieved, cleavage of the GST-tag will be performed by using the specific protease.

Finally, characterization of the intrinsic factor protein is going to be achieved. The characterization process involves the binding of vitamin B12 to the intrinsic factor protein.

Result

For purification and characterization of recombinant GIF protein, GIF gene has been amplified via PCR with primers that are designed with BamHI and XhoI restriction recognition sites. Amplified gene products and PGEX-6P1 were cut with BamHI and XhoI. Then, a ligation reaction was set up for GIF and vector. After transformation of ligated products, plasmids were isolated and were cut with BamHI and XhoI for validation (Figure 1).

Figure 1: Agarose gel result of BamHI and XhoI double cut of plasmid isolated from colonies which are transformed with ligation product

Module 3 is going to make a standard part which is crucially important for our whole experimental design in iGEM project. Project is basically about releasing of intrinsic factor (by GIF gene) in right part of the body and module 3 aims to activate GIF gene to maximum level by pH change in that part. Sensing pH difference is going to be made with pH-dependent promoter (nhaA promoter) that is designed in 2008 by another iGEM team called NYMU-Taipei (Figure 1). (1)

NhaA is responsible gene for Na+/H+ antiporter that has a promoter which can be defined as pH sensor.(2)

In our design, nhaA promoter is going to be inserted into our part in order to activate expression of OmpA+GIF as shown in Figure 2. NhaA promoter is going to be obtained by PCR technique.

Primers were designed by depending on promoter regions, which were defined as previous researches.(3) After all primer designing, the expected PCR product which consists of 192 bases, is shown in Figure 3.

pH dependency of promoter remote the releasing different amount of intrinsic factor in different pHs. The maximum releasing of intrinsic factor ought to be at pH 8.5. (1) It is also known that small intestine has alkaline pH.

After constructing that standard part, intrinsic factor protein is going to be isolated from extracellular medium to prove that protein of interest can be released to ileum.

REFERENCES

1.NYMU-Taipei. (2008). Team:NYMU-Taipei/Project/pH Sensor. Retrieved September 2, 2013, from iGEM 2008: https://2008.igem.org/Team:NYMU-Taipei/Project/pH_Sensor

2.Padan, E., Tzubery, T., Herz, K., Kozachkov, L., Rimon, A., Galili, L. (2004). NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter. Biochimica et Biophysica Acta, 2 –13.

3.Dover, N. and Padan, E. (2001). Transcription of nhaA, the Main Na+/H+ Antiporter of Escherichia coli, Is Regulated by Na+ and Growth Phase. Journal of Bacteriology, 644-653.

Result

DH5α template isolation for pcr purification of nhaA promoter. The left part of DNA MIX MARKER is done by manuel and the right part of marker is isolated by DNA isolation kit supplied by Anatolia Genework. For the next step, we used DNA template isolated by kit because of their concentration view on gel.

Figure 1: Agarose gel results of DNA isolation from DH5α strain

We have done gradient pcr between 55-62 oC annealing temperatures (first well is 55 and the last well is 62). As a result, we saw only one fragment at fifth (59.2 oC) well. We designed our pcr for 59 oC annealing temperature.

Figure 2: Agarose gel results of amplification nhaA promoter from DH5α strain via gradient PCR

PCR products of nhaA promoter at 59.2 oC annealing temperature. First well is a negative control (without template), second and third well shows us non-purified promoter fragments. Last well shows purified promoter fragment. We agreed that is our promoter of interest because according to sequence of nhaA promoter, it has 194 bp. It can be seen that fragment is close to 200 bp according to marker.

Figure 3: PCR amplification of nhaA promoter at 59 0C annealing temparature

Overview

Kill switch system is required to prevent the release of genetically modified organisms to the environment. Also, our genetically modified bacteria would be colonized in human intestine so we have to prevent the spread of GMO to the other part. To realize this aim, we benefit from glucose and auto-inducer 2 (AI-2) concentrations. MazE and MazF are stress-induced molecules that mediate cell death and their production would be controlled with two different promoters that sense glucose and AI-2. Furthermore, MazE is anti-toxic and MazF is toxic for the cell so they must be found in equilibrium. If the amount of MazF is more than MazE, the cell would die because of its toxic effects. Based on these reasons, our kill switch system consists of two parts.

How does it work?

The first part is just to keep alive in the presence of glucose. MazE would be produced by the sense of glucose and a weak constitutive promoter (BBa_J23102) is used for MazF production. Thus, MazF would be found in equilibrium with MazE. In the absence of glucose, amount of MazF would be more than MazE so cell would go to death.

Toxin-Antitoxin Module: MazE-MazF

Figure 1: The multiple levels of autoregulation of the MazF–MazE toxin–antitoxin system.

a | MazE and MazF are synthesized from the same promoter. MazE forms a complex with MazF under normal growth conditions. b | Under stress conditions, MazE is subjected to cleavage by an ATP-dependent protease. As released MazF cleaves its own mRNA, the expression of MazF is autoregulated. MazF activity leads to the arrest of bacterial cell growth and to eventual cell death. It has been shown that a few mRNAs are resistant to MazF in E. Coli.

The second part is to prevent the overproduction of cells. A promoter that sense auto inducible 2 molecules is used to control MazF production. If cells reproduce more, AI-2 is produced by cells and this promoter would sense AI-2, so MazF production would be increased. (Engelberg-Kulka, Hazan et al. 2005) Hence, MazE and MazF equilibrium would be broken and cell would go to death.

Structure

References

1. Engelberg-Kulka, H., et al. (2005). "mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria." J Cell Sci 118(Pt 19): 4327-4332.

2. Xue, T., et al. (2009). "LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing." Cell Res 19(11): 1258-1268.

3. Yamaguchi, Y., & Inouye, M. (2011). Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nature Reviews Microbiology. doi:10.1038/nrmicro2651

Result

Marker, Kan BB, PTS p0+1I, Amp BB, MazE+4F, and 12N+4F were respectively loaded into agarose gel.

Figure 1: EcoRI cut of first ligation reaction products

Kan BB, PTS p0+1I, Amp BB, and MazE+4F bands were expected, but gene fragments were not seen gel although there are large amount. 12N+4F was seen three bands, therefore; containing 12N+4F plasmid did not cut with restriction enzymes.

Marker, uncut, one cut , double cut 12N(RFP) and 1I(RBS) were respectively loaded into agarose gel.

Figure 2: Validation of ligation reaction

All bands are as expected. We understood that 12N and 1I were cut.