Construction Overview

ACHIEVEMENTS FROM OUR MAIN CONSTRUCTION:

We have successfully cloned SpPCS in pSB1C3 (BBa_K1526000) into E.coli DH5α

We have successfully cloned the intermediate construct pYodA + SpPCS in pSB1C3 (BBa_K1526004) into E.coli DH5α

We have successfully cloned Gsh1* (BBa_K1526001) in pSB1C3 into E.coli DH5α

We have successfully cloned CysP in pSB1C3 (BBa_K1526003) into E.coli DH5α

Characterizing the parts:

Creating the regulator LacRS

In order to characterize the parts we have created a new BioBrick compatible with our chassis. As E.coli DH5α does not have a lac repressor system we have assembled together basic parts J32100, B0033, C0012, B0010, B0012 and R0010 to create the composite regulator Lac Repressor System (LacRS)

Our first functionalization test coupling LacRS upstream the Green Fluorescent Protein (GFP) Biobrick (E0040) showed expression of GFP under UV light when the system is previously induced by IPTG.

We have successfully cloned and tested the functionality of LacRS in pSB1A3 (BBa_K1526009) into E.coli DH5α

Creating Inducible monofunctional constructs:

Each gene from our project was assembled downstream our LacRS Biobrick in order to allow a better characterization of the parts.

• We have successfully cloned LacRS+Gsh1* in pSB1C3 (BBa_K1526005) into E.coli DH5α

• We have successfully cloned LacRS+SpPCS in pSB1C3 (BBa_K1526006) into E.coli DH5α

• We have successfully cloned LacRS+MntH (BBa_K1526007) in pSB1C3 into E.coli DH5α

• We have successfully cloned LacRS+CysP in pSB1C3 (BBa_K1526008) into E.coli DH5α

Characterising CysP:

In order to characterise the sulfate secondary transporter from Bacillus subtilis we have acquired E. coli BW25113 strain mutants from the Keio collection for complementation assays.

We have used CysW (GenoBase code: JW2416) and CysA (GenoBase code: JW2415) mutants from the Keio collection as they represent deletions of different subunits (respectively, permease and ATPase) of the main sulfate transporter in E. coli (ABC transporter).

We have transformed both the mutants and the wild type with our LacRS+CysP plasmid and put them to grow on M9 media (1mM of Sulfate and 0.4%w/v of glucose) .

We still did not have time to make the optical density measurements on a 96-well plate on the FlouStar Omega plate reader kindly loaned to our team by BMG.

Improving the characterization of pYodA

pYodA (also called ZinTp) is a cadmium sensitive promoter endogenous to E. coli, where it regulates the bacterial response to stress stimulus after exposure to metal ions. As our final construct relies strongly on the sensing of cadmium we wanted to improve the characterisation of BioBrick BBa_K896008 submitted in 2012 by the NYMU Taiwan team.

We assembled the Green Fluorescence Protein GFP (BBa_E0240) under PYoda in order to assess the strength of promoter in response to different concentrations of Cadmium chloride (CdCl2) and its specificity to this metal.

First test: E. coli DH5A viability assay

To characterise PYoda we adjusted the OD600 of an E.coli DH5alpha overnight transformed with pSB3K3 carrying BioBrick to 0.1 in LB broth.

The same procedure was done to an overnight culture of E. coli DH5 alpha (WT) and to a DH5 alpha transformed with pSB3K3 carrying GFP under the control of the constitutive promoter BBa_I20260 (ConstGFP). These would be our negative and positive control in fluorescence expression respectively.

Then, different concentrations of CdCl2 was added to the cultures with adjusted OD600 and 200uL were transferred onto a 96 well plate (Diagram 1.) and incubated overnight at 37ºC in a plate reader.

Fig 1. Distribution of DH5alpha carrying GFP under BBa_I20260 (ConsGFP); untransformed DH5alpha (WT) and DH5alpha carrying GFP under PYoda (PYodaGFP) in the 96 well plate. Different concentrations of Cadmium chloride (CdCl2) were assessed ranging from 0.1 to 0.9 mM. LB broth was used as blank after testing no significant changes in OD600 absorbance upon Cadmium concentration.

We measured the absorbance overnight of the different mutants using LB broth as blank -after assessing that Cadmium chloride did not affect absorbance-. The concentration of Cadmium reflects a negative effect around 0.9 mM concentration in all mutants, being pYoda the best growing mutant. However, the wild type presents depleted growth compared to the mutants in absence of CdCl2.

Figure 2. Absorbance of DH5alpha (WT); mutant carrying GFP under Pyoda (pYoda) and mutant carrying GFP under constitutive promoter BBa_I20260 (pCons). Mutants were incubated at 37ºC with different CdCl2 concentrations (0 - 0.9mM) in the plate reader and measurements were taken every 20 minutes overnight.

Second test: Inducing pYodA with different cadmium concentrations and assessing its strength by using GFP expression as a proxy

The test was performed using 96 well plates arranged as shown in diagram 1. The results (not shown) did not present inducibility of PYoda regardless of the Cd concentration and fluorescence levels remained under the spectra of the wild type’s. This could suggest our chassis (Dh5Alpha) may not have the necessary apparatus for the induction of PyodA as it is known that the induction of the promoter by the heavy metal is not direct. It is mediated by many different genes related to facing stressful conditions in E. coli such as Soxs and Fur (Puškárová, A., et al., 2002).

(Puškárová, A., et al. "Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli." Microbiology 148.12 (2002): 3801-3811.)

Due to the time restrictions at the time of the wiki freeze we haven not optimised the characterisation of PYoda to the standards that we expected. However, we are already working on an alternative chassis, the E. coli bw25113 strain, in which we will assess the response of Cadmium.

Third Test: Try inducing PyodA with other metals to test its efficiency as a cadmium biosensor.

As a collaboration and because we are helping to the synthetic phytochelatin characterisation we have asked the Imperial College team to characterise the pYodA inducibility to metal ions other than Cadmium. We are hoping to get some results before going Boston in order to address the question on how specific pYodA is to Cadmium.