Team:Glasgow/Demonstrate

Glasgow iGEM 2017

Demonstrate

Overview

During the time working on CampyLOCATOR, we carried out many individual sub-projects which, combined, form our biosensing device. Due to time limitations we were unable to get as far as combining them - however, we did showcase working parts of our project on this wiki which could be improved upon with more time. This page contains short descriptions of the work we did demonstrating the functionality of our sub-projects, with links to the pages with longer descriptions if these are of interest.

Mannitol Operon

In the mannitol operon sub-project, we aimed to test an inducible promoter system (mtlR-mtlE) taken from the mannitol operon of Pseudomonas fluorescens. We tested the induction of the linked promoter (PmtlE) with various sugars in order to replicate results from Liu et al (2015).^[1] We produced the following graph:

Figure 1: Plasmids, code names and testing.The top image shows our construct, the part names and their code names. The graph shows OD normalised fluorescence minus L-broth background for each sugar condition. The variant construct plasmids were all tested. The OD reflects the fluorescence of each construct tested in the presence of sugar. Again, ribose and sorbitol show the greatest fluorescence response across the plasmids with the regulatory plus reporter plasmid showing the overall greatest fluorescence levels. The regulatory constructs are in pSB1C3 whilst the reporter plasmids are in PSB3K3. DH5α was used as a control. The bottom table shows the plasmid names and their code names.

The results obtained from our tests do not reflect the findings of Liu et al ^[1]. We got positive results for the working of the regulatory protein, mtlR and its regulated promoter, PmtlE in E. coli in response to ribose and sorbitol. Whilst Liu et al demonstrated a strong response to sorbitol and mannitol, our constructs showed ribose and sorbitol evoke the largest fluorescence response across all plasmids. A potential reason our results do not reflect those of Liu et al is the use of different organisms. Our findings could show that E. coli exhibits a different response to ribose and mannitol than Pseudomonas fluorescens. E. coli may not utilize these sugars in induction of this system and therefore downstream genes are not turned on as they are in Pseudomonas.

Further detail on this sub-project can be found on the mannitol operon page of our wiki.

Mutagenesis of the L-Arabinose Operon

Another sub-project we worked on was mutagenesis of the L-arabinose operon as an alternative route to biosense our target sugar, xylulose. In this sub-project we constructed new BioBricks, splitting p_BAD and AraC into two seperate BioBricks instead of being combined in the case of BBa_I0500. We showed that these BioBricks were functional in seperate plasmids, and additionally showed successful mutagenesis of the AraC protein in order to alter its characteristics. Future iGEM teams working with the detection of small molecules will be able to work with these tools to create new biosensors.

Further detail on this sub-project can be found on the Arabinose Operon Mutagenesis page of our wiki.

Xylulose Biosynthesis

Our xylulose biosynthesis sub-project had a focus on production of xylulose, as we found that costs were too high to do testing of our constructs with this sugar. We carried this out through overproduction of an enzyme named xylose isomerase, which converts xylose into xylulose in many organisms. We utilised the T7 promoter system in order to do this.

Figure 2: Expression Plasmid Assembly and Testing: A) Protein expression analysis compared three BL21 (DE3) colonies before (Pre) and after (Post) construct expression was induced with IPTG. Marker used was 1kb+ ladder (Invitrogen) B) Gel electrophoresis of the L18 (Ligation 18) expression plasmid after restriction digestion shows the insert and vector separate, and with expected band sizes.

In all induced cultures, a stronger band of ~50kD appears when compared to uninduced cells. This band size corresponds to the predicted molecular weight of the xylose isomerase protein (49.742kD, calculated by EMBOSS Pepstats) from E. coli. This clearly suggests that xylose isomerase is being overexpressed by the expression plasmid.

Further detail on this sub-project can be found on the Xylulose Biosynthesis page of our wiki.

Engineering

References

↑ ^1.0 ^1.1 Liu, Y., Rainey, P., and Zhang, X. (2015). Molecular mechanisms of xylose utilization by Pseudomonas fluorescens: overlapping genetic responses to xylose, xylulose, ribose and mannitol.

[Liu-1] 1.0 ^1.1 Liu, Y., Rainey, P., and Zhang, X. (2015). Molecular mechanisms of xylose utilization by Pseudomonas fluorescens: overlapping genetic responses to xylose, xylulose, ribose and mannitol.

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