Team:Peking/Project/Bioabsorbent/InductiveAggregation

From 2010.igem.org

Revision as of 20:00, 27 October 2010

Inductive Aggregation

Our bioabsorbent works as expected now. However, there is still a problem remained in the application of our bioabsorbent. It is not convenient to simply remove our bacteria from water or gather up them for recycling process after enough Hg (II) has been absorbed. Therefore, a time-delayed autoaggregation design was introduced. We chose Antigen43 (Ag43), which mediates visible autoaggregation conferred on the bacteria population in static liquid [1], as our autoaggregation effector.

Antigen 43 is a unique autotransporter that promotes bacterial cell-to-cell aggregation. This protein, antigen 43(Ag43), is encoded by a single gene originally identified in 1980 and designated flu in relation to the aggregative property antigen 43 expression confers upon the cells. Antigen 43 can be expressed on the E.coli cell surface in a large copy number, up to 50000 copies per cell, resulting in characteristically frizzy colony morphology [2].

The structure analysis of antigen 43 revealed that antigen 43 possesses the typical autotransporter protein domain: an N-terminal signal peptide; an N-proximal passenger domain that is secreted, which could also be called α domain; an autochaperone domain that facilitates folding of the passenger domain; and a C-terminal β-barrel domain that forms an integral outer membrane protein, also called β domain[6], as is shown in Fig 1. The 52-amino-acid signal peptide of the antigen 43 protein consists of a C-terminal domain that resembles a classic signal peptide and an N-terminal extension that is conserved among many members of the autotransporter family. The passenger domain(αdomain) confers the autoaggregation phenotype. Theαdomain which has pronounced surface expression, is bound to the surface via non-covalent interaction with the βdomain and can be selectively detached from the outer membrane by brief heating to 60℃. Like the passenger domain of all members of the autotransporter family, theαdomain is predicted to form a β-helix, which in antigen 43 consists of 18 rungs of 16-19 amino acids [6]. However, some conserved motifs that are found in many other passenger domains, which are involved in protein-protein interaction or to facilitate binding to host cells, are not functional in theαdomain of antigen 43, indicating that antigen 43 may have other ways to mediate cell-cell interaction. βdomain, on the other hand, is shown to consist of the integral outer membrane translocator domain and a surface-exposed βhelical structure. It is a heat-modifiable integral outer-membrane protein (Fig 1).

Figure 1: Schematic showing the structure of the coding sequence of antigen 43. Antigen 43 contains a signal peptide (red), a passenger domain (green), an autochaperone domain (blue) and a translocation unit (light blue), which are all indicated in this figure. Adapted from [2].

Antigen-43 mediated aggregation is a distinct phenotype that can be visualized macroscopically as flocculation and settling of cells in static liquid suspensions. In immunofluorescence studies of antigen 43-producing strain, the protein can be visualized as evenly distributed over the surface of the entire cell. As a result, the motility of bacteria will be restrained due to the expression of antigen 43.

Construction

Since we engineered E.coli to detect and absorb heavy metal ions, it is of great importance that we consider the disposal. Generally, our biosensor and bioabsorbent are based on the principle of application ease, which requires minimal effort remove the bacteria.

When designing our heavy metal decontamination kit, we must take horizontal gene transfer into consideration, which is also our main topic of human practice. Horizontal gene transfer is defined as the movement of genetic material between bacteria other than by descent, in which genetic information travels through the generations as the cell divides, and it has always been a problem that challenges use of engineered bacteria. The most effective way to avoid horizontal gene transfer is to collect the bacteria after use before carelessly discarding them. In consistent with this, Antigen 43 was brought into use. When antigen 43 is expressed, the bacteria will aggregate in liquid at population level. After nearly 20 minutes, the OD value of the supernatant will decrease to nearly 0.05, indicating that few bacteria were left in the supernatant. In contrast to the control, bacteria that express antigen 43 showed dramatically OD value decrease only after 20 minutes, which shows the powerful function of antigen 43 for actual application.

Moreover, as is discussed above, our bioabsorbent functions to decontaminate Hg (II) to a non-toxic concentration via binding Hg (II), including cytoplasmically expressed MBP, periplasmic ally expressed DsbA-MBP and surface displayed Lpp-OmpA-MBD. However, Agn 43 shall not be expressed immediately when Hg (II) emerges. Instead, a time delay is necessary to guarantee the metal absorption efficacy. Hence, a time-delay device should be introduced. This is achieved by construction of a genetic cascade, as is shown in Fig 2.

With this time-delayed device, the expression of Ag43 will be in response to the presence of mercury with a time delay. Besides time-delayed function, this design also acts an amplifier that can promote the expression level of antigen 43 as well.

Materials and methods:

In order to clone antigen 43 into standard plasmid, we first obtained its coding sequence from the genome of E.Coli K12 strain using nest PCR. Primers were designed using Primer Premier 5 and with standard restriction sites, EcoRI, XbaI for the forward and SpeI, PstI for the reverse. This gene represented an apparent band of 3kb in size when electrophoresis was conducted in 1% agarose gel. Then this fragment was cloned into pSB1C3.

Figure 3：Gel image of the final cycle of PCR for antigen 43. Lane 3 is Trans2K DNA marker. Other 4 lanes was Antigen 43 in size of about 3kb large.

Other components shown in Fig 2 were assembled with Antigen 43 step by step with Standard assembly. Particularly, 6 PstI restriction sites in the coding region of Agn 43 were removed by synonymous mutations.

We adapted a well-established auto-aggregation assay to test the function of antigen 43[1]. This assay is to follow the bacterial settling kinetics over time. BL21 strains bearing Agn 43 were overnight cultured and were diluted 1:100 in 50ml LB and then grown to OD600=0.4-0.6., at which point expression of antigen 43 was induced by the addition 0.001% IPTG. The cultures were grown to a final OD600=1 (standardized) and 5000rp for 5min, then use 1% PBS which 0.15mM NaCl was added to resuspend it. This culture was vigorously shaken before experiment. At regular time interval, a 100ul samples was taken approximately 0.5cm from the surface and transferred into a microplate maintained on ice. Every 10 minutes 100ul sample was taken with same method. At the end of the experiment, OD600 were measured using microplate reader. At last the plot was drawn by OD600 with time.

Result

We induced the expression of antigen 43 by IPTG. After 4 hours we placed the tube on the table to see the auto-aggregation process. After 20 minutes there was significant aggregation which can be easily observed by naked eyes, which was recorded by digital image.

Fig 4: Autoaggregation mediated by antigen 43. The left is strain with blank plasmid, and the right is strain expressing antigen 43, which showed significant autoaggregation.

Then the result of the auto-aggregation assay is shown in Fig 5. The strain bearing Agn43 but was not treated with IPTG also showed significant drop in OD600. We speculate that this was because the leakage expression of T7 polymerase in BL21 strains and the amplifier effect of the time delay device.

FIgure 5: The OD value of the supernatant varying with time. There was a significant drop of OD value after nearly 20 minutes in the strain inductively expressing antigen 43. Then blank strain do not express antigen 43 showed constant OD value of the supernatant during the whole process.

In summary, the inductive aggregation module engineered from Agn43 worked as expected, which means that bacteria will automatically aggregate and can be easily removed from water after the bioabsorbent completes its task.

Reference:

1. Per Klemm, Louise H( 2005). Molecular Microbiology. 51(1),283-296.

2. Ian R.Henderson, Mary M(1997). FEMS Microbiology Letters 149.115-120.

3. Kristian Kjargaard, Henrik H(2002). Journal of Bacteriology. 184.15.4197-4204.

4. Marjan W, Ian R.Henderson(2008). Annu.Rev.Microbiol. 62:153-169.

5. Henrik H, Trinad Chakraborty(1999).Journal of Bacteriology. 4834-4841.

6. Andrew J.Roche, Joanna P.McFadden(2001).Microbiology.147,161-169.

7. Orla Sherlock, Ulrich Dobrindt(2006). Journal of Bacteriology. 188.5.1798-1807.