Difference between revisions of "Part:BBa K2442202"

Latest revision as of 01:33, 2 November 2017

Ptet + RBS + MtlR

This composite part contains the mtlR coding sequence along with the ptet promoter. mtlR coding sequence is derived from the mannitol operon from Pseudomonas fluorescens. The ptet promoter is a well characterised promoter part R0040. This composite Biobrick was used in conjunction with BBa_K2442203. By splitting the regulatory protein (mtlR) from the promoter it regulates (pmtle) allowed for greater control over the expression of GFP.

Usage and Biology

The mtlR regulatory protein is found naturally in Pseudomonas fluorescens. The mtlR protein has been described as a multi-sugar transcriptional regulator. There is some evidence to suggest that the protein functions as a dimer. Naturally this multi-sugar regulator functions to switch on sugar metabolism genes in P. flourescens in the presence of organic sugars.

The aim of our project was to harness the natural regulatory abilities of the mtlR protein and use this alongside the promoter that it regulates (pmtle). We also aimed to combine this regulatory system with a reporter construct BBa_K2442203 to evoke GFP response.

We used the sequence of the P. flourescens mtlR gene to construct a coding sequence that was regulated upstream by the ptet promoter. By co-transforming E. coli cells with BBa_K2442202 and BBa_K2442203 we were able to create a sugar responsive assay that produced GFP via induction by sugars.

Characterisation

We constructed two plasmid constructs - the regulatory plasmid BBa_K2442202 responsible for the transcription and translation of the MtlR protein, and a separate reporter plasmid which would allow for a measurable response of MtlR-xylulose interaction. mtlR coding sequence was ordered for synthesis as a G-block construct from IDT and was ligated into pSB1C3. As this was to be under the control of a tet promoter, the tet promoter and medium RBS B0032 were ordered as oligos from IDT and were ligated into a separate pSB1C3 plasmid. In order for the mtlR sequence to be behind the promoter and RBS, mtlR was restriction digested from its plasmid and ligated into the promoter plasmid. This produced our final regulatory plasmid.

The reporter plasmid was constructed by first ligating the mtlE promoter into pSB1C3. We ordered the sequence Liu et al (2015) [1] used, which contained the native promoter and RBS. We also decided to experiment with a variety of RBS in order to find the optimal one for our genetic circuit. We used the male promoter ligated to two different RBS, one stronger (B0034) and one weaker (B0032). Overall we created three reporter constructs, the wild type reporter plasmid (BBa_K2442203), and two variants, BBa_K2442204 and BBa_K2442205. These variants were ordered as single-stranded oligonucleotides, annealed together (to make double-stranded oligonucleotides) and then ligated into pSB1C3. We then used GFP from the kitplate (E0040). This was restriction digested out of the plasmid and ligated behind Ptet and the various RBS.

In order to test in the presence of both plasmids, the gene construct present in the reporter plasmid was built in pSB1C3 and then digested and ligated into a new plasmid, pSB3K3. This ensured both plasmids had a different origin of replication and therefore could both be present in one cell. It also ensured we could select for cells which contained both the plasmids required to carry out testing.

Sugars were selected based on previous work with the mannitol operon and other similar sugars available in the lab were used. We tested these combinations with 6 different sugars; mannitol, sorbitol, arabinose, xylose, ribose, and fructose. We could not test xylulose due to its prohibitive cost, which is discussed on the [http://2017.igem.org/Team:Glasgow/XyluloseBiosynthesis Xylulose Biosynthesis] subproject page of our wiki. Our controls were DH5α empty cells, fresh LB, and each plasmid alone. The two constructs were individually tested with a variety of sugars in order to record the fluorescence in the absence of MtlR. Controls were set up without the constructs in order to measure the basal level of GFP fluorescence.

Figure 1: 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.

Our results showed a distinct GFP fluorescence response to the sugars ribose and sorbitol, in cells that carried both the regulator and reporter constructs. A lower response to sugar induction is observed in E. coli cells containing only the reporter construct, with no mtlR. This shows that the PmtlE promoter has some sugar induced functionality in E. coli, but that there is also an increased response in the presence of its mtlR regulator partner. This result confirms the findings from P. flourescens that mtlR acts as a transcriptional activator of the PmtlE promoter, when the circuit is transferred into an E. coli cell background. The different pattern of sugar-activation requires further study, and may also be due to the effect of a different cellular background.

Sequence and Features