Coding

Part:BBa_K519010

Designed by: Kotone Miyake   Group: iGEM11_Tokyo-NoKoGen   (2011-09-30)

SmtA

SmtA is a metallothionein that can bind to heavy metal ions such as Cd(II). This SmtA is cloned from Synechococcus sp. PCC7942, a cyanobacterial strain. SmtA is a type II metallothionein with a reduced cys content.It was reported that marine cyanobacterium that does not originally harbor a homologous gene, when genetically engineered to express SmtA, could tolerate medium containing Cd(II), Zn(II) and Cu(II) (Sode et al., 1998).


Metallothionein2.jpg

The data above, taken from a paper (Sode et al., 1988), shows that a cyanobacterial strain was able to tolerate medium of high Cd(II) concentration when it harbored the smtA gene.


For our iGEM project this year, we have successfully observed that SmtA, expressed under a constitutive promoter (J23109) work very well. We plotted a graph of growth rate by looking at the OD595 every 1 hour, and compared the growth of wild type E. coli and SmtA expressing E. coli. As a result, we found that E. coli expressing SmtA grow better than the WT E. coli at a high Cd(II) concentration.


SCAU-China 2021 iGEM TEAM

SmtA contains four zinc ions. The protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn4Cys9His2 cluster. The four Zn2+ ions and five bridging Cys thiolate sulfurs form two fused six-membered rings with distorted boat conformations [1]. SmtA sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA [2].

Moreover, one of the two Cys coordination zinc ions in SmtA is easy to exchange with the exogenous metal (cadmium)(Fig.1), and the other is inert and hard to replace.


T--SCAU-China--K4014006.jpg

Fig.1. Proton-decoupled one-dimensional 111Cd NMR spectra of SmtA.


111Cd4SmtA prepared by reconstitution of apo-SmtA with 111Cd2+ (106.04 MHz,pH 7.0, 318 K, 10% D2O, 50 mM TriszHCl, 50 mM NaCl) showing four peaks with equal integrals. (b) Substitution of Zn2+ by Cd2+. Spectra recorded after mixing Zn4SmtA with 1 (bottom), 4 (middle), and 8 (top) mol equivalents of 111Cd2+ (pH7.0, 308 K, 10% D2O, 50 mM TriszHCl, 50 mM NaCl). Comparison with a shows that the first mol equivalent of 111Cd2+ selectively occupies binding site B, and that binding site A is not occupied at all. The various peaks reflect 111Cd2+ in mixed Cd, Zn clusters. The occupation of the sites is depicted by squares and circles in the order ABDC, clockwise from top left. Filled square, Cd in the site which gives rise to the respective peak; filled circle, Cd in an adjacent site; open circle, Zn; e.g., the peak at 656 ppm corresponds to111Cd2+ in site B, with Zn2+ in sites A and C and 111Cd2+ in site D. (c) Metal-to-ligand connectivities for SmtA (as deter-mined by 2D heteronuclear NMR experiments) [1].

In addition, SCAU-China also optimized the codon according to yeast codon bias. The experimental results are shown in BBa_K4014012

We have improved this part, more details please see http://parts.igem.org/Part:BBa_K4014012

Edited by SCAU-China 2021.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 93
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 93
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 93
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 93
    Illegal AgeI site found at 121
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

function-NA-
n/aSmtA

Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden Value: -0.1 ± 3.9%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

[edit]
Categories
//binding/metal
//proteindomain
Parameters
function
n/aSmtA