Part:BBa_K1058008:Experience

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•• iGEM Dundee 2015

This part was ordered from the 2013 submission plates and sequenced by iGEM Dundee 2015 and found to be correct. The part was tested as a stand-alone device by iGEM Dundee 2015 and also deconstructed into a new promoter biobrick (BBa_K1590003) and a repressor (chrB) biobrick (BBa_K1590004). Dundee iGEM 2015 found this part to be partially functional but in need of further optimization.

Group: CSI:DUNDEE, University of Dundee iGEM team 2015 Author: Manuel Blank

Summary: iGEM Dundee requested this part from the registry with the goal of building a chromate sensor that will be able to detect traces of stainless steel in biological samples. This was intended as a key component of of forensic toolkit.

The BBa_K1058008 biobrick was a complete device with eGFP hooked to a promoter and repressor gene from Ochrobactrum tritici 5bvl1. iGEM Dundee initially sequenced this brick in its entirety, and found the sequence to match perfectly that deposited here. Next, iGEM Dundee set out to test the functionality of the BBa_K1058008 part in the E. coli chassis MG1655 (Figure 1). Western analysis showed production of GFP in the absence of additional chromate.

Figure 1: Comparison of presence of GFP in MG1655 + BBa_K1058008, MG1655 + pSB1C3-Pchr-gfp (A) + pUniprom-chrB, and MG1655 + pSB1C3-Pchr-gfp (A) + pUniprom-chrB (opt).

Next, iGEM Dundee cloned the separate chr promoter and chrB repressor using RFC[10] intp pSB1C3 resulting in new biobricks BBa_K1590003 and No part name specified with partinfo tag., respectively. The functionality of the chr promoter was tested by again hooking up GFP (Figure 2).

Figure 2. Western analysis of GFP production driven by the chr promoter: Single colonies of JM110 + pSB1C3-Pchr-gfp (A) and MC1061 + pSB1C3-Pchr-gfp (B) were used to inoculate 5 ml of LB broth supplemented with 100 ^µg/_ml chloramphenicol. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 1 ml TBS. 100 µl of the sample was mixed with 100 µl laemmli buffer, and boiled for 10min. 3 µl of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and PmanA-gfp as a positive control.

The biosynthesis of ChrB in E. coli was characterized by attachment of a hexa-histidine tag and hooking up to the constiutive Tat promoter (BBa_K895000 in the AmpR vector pUNI-PROM (Figure 3). The hexa-histidine tag allows Western analysis with an anti-His antibody (Figure 3).

Figure 3: Western analysis of ChrB production from the Tat promoter BBa_K895000: Single colonies of JM110 pUniprom-chrB and JM110 pUniprom-chrB (opt) were used to inoculate 5 ml of LB broth supplemented with 100 ^µg/_ml ampicillin. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 200 µl laemmli buffer, and the sample boiled for 10min. 10 µl of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and hydA - 6-His as a positive control. Expected sizes: GFP ~ 27kDa, ChrB ~35kDa.

On conclusion, iGEM Dundee found that the chr promoter within the original BBa_K1058008 biobrick may already de-repressed when placed in an E. coli chassis.

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