Part:BBa_K346007

Antigen 43

Introduction:

Antigen 43 is a unique autotransporter that promote 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(αdomain) confers the autoaggregation phenotype and it is bound to the surface via non-covalent interaction with the βdomain.

Figure 1: The structure of the coding sequence of antigen 43. Antigen 43 contains a signal peptide, a passenger domain, an autochaperone domain and a translocation unit, which are all indicated in this figure.

Antigen 43 mediated aggregation is a distinct phenotype that can be visualized macroscopically as flocculation and settling of cells in static liquid suspensions. Since we use E.coli to detect and absorb heavy metal ions, it is of great importance that we consider the disposal,which requires minimal effort to reclaim the bacteria, thus purifying the polluted water.

Materials and methods:

1: construction of standard plasmid with antigen 43 gene: in order to put antigen 43 gene into standard plasmid, we first cloned it from the genome of K12 strain of E.Coli using nest PCR procedure. Primers were designed using Primer Premier 5 and with standard digest sites, EcoRI,XbaI forward and SpeI, PstI reverse. This gene showed an apparent 3K band when electrophoresis in 1% agarose gel in both two steps of PCR. This gene is then put into standard plasmid by standard digestion.

Figure 2：the gel image of antigen 43. The middle is Trans2K DNA marker. Antigen 43 is about 3k large.

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, 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 clone plasmid that can achieve 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：function analysis of antigen 43: Because antigen 43 can cause auto-aggregation, we adapted a well-established auto-aggregation assay to test the function of antigen 43[4]. This assay is to follow the bacterial settling kinetics over time. BL21 strains 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 re-suspense it. this cultures were 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 samples 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 adding IPTG. After 4 hours we put the tube on the table to see the auto-aggregation phenomenon. After 20 minutes there is significant aggregation which can be easily observed by eyes. We then took photograph to show this phenomenon as figure 4.

Figure 4: Autoaggregation caused by antigen 43. The right one is strain expressing antigen 43, which shows significant autoaggregation.

Then the result of the auto-aggregation assay is shown in figure 5. The strain that did not add IPTG also shows significant drop in OD600. We propose this is because the leakage expression of T7 polymerase in BL21 strains and due to the amplifier the expression of antigen 43 is in great amount.

FIgure 5: The OD of the supernatant to time. There is a significant drop of OD after nearly 20 minutes in those strains that expressing antigen 43, and those do not express antigen 43 show constant OD of the supernatant.

Reference

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1.Marjan W.van der Woude & Ian R.Henderson(2008).Annu.Rev.Microbiol.62:153-169.

2.Kristian K & Henrik H(2002).Journal of Bacteriology.184.15.4197-4204.

3.Ian R.& Herderson(1997).FEMS Microbiology Letters.149.115-120.

4.Glen C.Ulett & Richard I.W(2006).Microbiology.152,2101-2110.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 709
  Illegal NgoMIV site found at 1933
  Illegal NgoMIV site found at 2443
  Illegal NgoMIV site found at 2464
  Illegal AgeI site found at 1459
  Illegal AgeI site found at 2203
  Illegal AgeI site found at 2617
  Illegal AgeI site found at 2859
  
• 1000
  COMPATIBLE WITH RFC[1000]