Difference between revisions of "Part:BBa K346007"

Latest revision as of 20:51, 4 October 2011

Antigen 43

Introduction:

Antigen 43 is a unique autotransporter that promotes bacterial cell-to-cell aggregation. Antigen 43 can be expressed on the E.coli cell surface in large quantities, up to 50000 copies per cell.[1] The structure analysis of antigen 43 revealed that antigen 43 has 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[2]. The passenger domain(α domain) confers the autoaggregation phenotype and it is bound to the surface via non-covalent interaction with the β domain.

Figure 1: Schematic figure for the structure of antigen 43's coding sequence. 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 [1].

Materials and methods:

1: 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 2：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.

2: Construction of the expression plasmid of antigen 43: in order to express antigen 43, we put in downstream the delay and amplify part which are mentioned before, which is constructed by PhiR73 and Po promoter. This PhiR73 is under T7 promoter's regulation, so antigen 43 gene is indirectly controlled by T7 promoter. We then put this plasmid into BL21 strains, and test the function of antigen 43 by IPTG induction.

Figure 3: The construction of the plasmid which can realize the function of antigen 43. Green circle indicate the ribosome binding sites. Red hexagon indicates the terminator. PhiR73 and Po promoter are used as a delay and amplify module.

3：We adapted a well-established auto-aggregation assay to test the function of antigen 43[1]. This assay is designed to trace 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. Finally 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.

Figure 4: Autoaggregation mediated by antigen 43. The left is the strain with blank plasmid, and the right is the 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.

Reference

[1] P Marguet, F Balagadde, C Tan, L You. Biology by design: reduction and synthesis of cellular components and behaviour. JR Soc Interface 4, 607–623 (2007). [2] CA Voigt. Genetic parts to program bacteria. Curr Opin Biotechnol 17, 548–557 (2006). [3] K Clancy, CA Voigt. Programming cells: towards an automated 'Genetic Compiler'. Curr Opin Biotechnol 21, 572–581 (2010) . [4] KA Egland, EP Greenberg. Conversion of the Vibrio fischeri transcriptional activator, LuxR, to a repressor. J. Bacteriol 182, 805-811 (2000). [5] M Schuster, ML Urbanowski, EP Greenberg. Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR. PNAS 45, 15833-15839 (2004).

Sequence and Features