Team:Sheffield/Parts

	BIOBRICKS

Introduction

Following our project we submitted 4 biobricks to iGEM 2016 registry. 2 of the biobricks included different hemerythrins genes: mc and td; one was an sfgfp gene under control of a strong promoter with an additional regulatory region operated by RyhB; the last biobricks was a modified strong promoter followed by a ribosomal binding site. Promoter sequences were modified using the iGEM biobrick Part: BBa_J23100. All submitted biobricks are summarised in the table below. Unfortunately we were unable to clone Dcr, medium promoter and Med-RyhB-GFP into our pSB1C3 plasmid, due to XbaI and SpeI restriction sites having the same sticky-ends, thus creating self-ligated plasmid on multiple occasions.

BBa K2016000	BBa K2016003
BBa K2016004	BBa K2016005

Strong constitutive promoter

During our project we have chosen 2 biobrick promoters (J23100 and J23106) and modified them to be suitable for direct cloning. Such an approach allows for amplification of the modified promoter without the need to carry out more cloning steps and modifications. We designed our biobricks as being composed of a working medium or strong promoter followed by a ribosomal binding site designed for E. coli strains. With such a design, anyone using our biobrick will be able to clone their construct directly in front of a desired gene and express their protein. Our biobricks were cloned into pSB1C3 plasmids as shown below. Due to difficulties with cloning, only the strong promoter was submitted to the registry (BBa_K2016000).

Figure 1: Map of BBa_K2016000 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites

Characterisation

We characterised our promoters by placing our sfGFP construct under the control of these promoters, and measuring their fluorescence as an indicator of gene expression.

Figure 2. Fluorescence of JC28 mutants or W3110 wild types transformed with RyhB-GFP constructs under the control of medium (MedGFP) or strong promoters (StrGFP).

McHr - hemerythrin

Methylococcus capsulatus hemerythrin (McHr) is a protein expressed by methane-oxidising bacterium: Methylococcus capsulatus. The family of hemerythrins can undergo a slow process of oxygenation upon binding of iron which causes their colour change from colourless into red/yellow. Its mechanism of oxygenation has been discovered to be much more rapid than other hemerythrins we investigated (DcrHr or TdHr). This gene was synthesised based on the GeneBank sequence entry and cloned into the pSB1C3 plasmid.

Figure 1: Map of BBa_K2016003 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites, however it is cloned in reverse orientation due to XbaI and SpeI enzymes having the same overhangs after restriction digest. To use this biobrick future teams should use PCR apmlification rather than restriction digest to clone this biobrick.

Characterisation

We believe we were able to express McHr in E.coli cells using a pET28a overexpression plasmid under the control of an IPTG inducible promoter, however its induction is not as clear as in the other bands indicating that it may need further characterisation (Fig. 2).

Figure 2. SDS-PAGE of expression of Dcr, Mc and Td in cells before and after induction with IPTG.For each protein 2 different biological repeats were first grown overnight and then induced with IPTG, L- ladder, U – uninduced, I – induced. For Mc (14.7 kDa) we can see induction of expression is less clear after adding IPTG, and should be investigated further.

TdHr - hemerythrin

TdHr is a protein expressed by Themiste dyscritum, a eukaryotic marine organism, in which this protein functions as an iron-binding transporter, much like our own haemoglobin. Td undergoes a slow process of oxygenation upon binding of iron and gains a violet/pink colour. This gene was codon-optimised for the use in E. coli and cloned into the pSB1C3 plasmid.

Figure 1: Map of BBa_K2016004 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites.

Characterisation

We were able to express TdHr in E.coli cells using a pET28a overexpression plasmid under the control of an IPTG inducible promoter (Fig. 2).

Figure 2. SDS-PAGE of expression of Dcr, Mc and Td in cells before and after induction with IPTG.For each protein 2 different biological repeats were first grown overnight and then induced with IPTG, L- ladder, U – uninduced, I – induced. For Td (13.5 kDa) we can see clear induction of expression after adding IPTG.

Str-RyhB-GFP - Superfolder GFP under the control of RyhB

Our superfolder GFP was cloned to be operated by RyhB on the RNA interference level. RyhB is responsive to iron concentrations within the cell and therefore provides us with a viable tool needed for iron detection. Strong or medium promoters were used for expression of gfp on different levels. However, due to difficulties with cloning, only the GFP under control of strong promoter was submitted to the iGEM registry. The gene was cloned into pSB1C3 vector as shown below.

Figure 1: Map of BBa_K2016005 biobrick cloned into pSC1C3 plasmid. Biobrick is cloned between XbaI and SpeI sites.

Characterisation

We demonstrated that our RhyB reporter system responds to increased iron concentrations by adding iron to our strains that had been transformed with our Str-RyhB-GFP and Med-RyhB-GFP constructs, after which we detected a significant increase in fluorescence

Figure 2. Change in GFP expression after 90 minutes following addition of FeCl₃ to a final concentration of 100 μM (A) or without addition of FeCl₃ (B). There is a significant increase in fluorescence upon addition of iron over the course of 90 minutes (A), however this was not seen over the same time period without addition of iron (B). Significance was assessed using a paired t-test and is conveyed with * <0.05, **<0.01, *** <0.001, **** <0.0001.