Revision as of 13:08, 30 October 2017

Our Results

During our project, we had three smaller teams within our main team. Each of these teams had different but interlinked objectives. Below we have outlined the main achievements of those projects:

Generation of novel Phytobricks

We successful generated a range of phytobricks that have been submitted to the registry and can be utilised by future iGEM teams who are interested in using the plant expression systems in their projects. Details of these phytobricks can be found on our Basic and Composite parts pages.

Generation of a new molecular tool to test the efficacy of tobacco inflitration

Team Luciferase created the 35S:Luc+:NosT construct , and quantified the activity of the 35S promoter using a luciferase reporter assay. These assays are detailed on the Part Experience page.

This image shows leaf discs taken from plants infiltrated with Agrobacterium containing the 35S:Luc+:NosT construct. The red regions show high levels of luciferase expression. This part can now be used by other teams in the future as a control when performing luciferase reporter assays.

Team TSH

Team TSH was responsible for creating constructs containing TSH or TSHH (TSH antagonist with His-tags for purification), controlled by the LexA promoter. This is part BBa_K2404016 . They also attempted to create a part with 35S:TSHH:NosT, but these ligations failed. Using their TSHH constructs, they infiltrated N. benthamiana and performed a protein extract which was ran on a protein gel to identify the presence of the antagonist.

This photo shows the DNA gel that showed the level 1 reaction to create the LexA:TSHH:NosT construct had worked. The DNA solution that this was taken from was later sequenced to confirm that the construct was correct.

This image shows a protein gel that team TSH created to test whether part BBa_K2404016 had been expressed in N. benthamiana . There was no band present where we anticipated, so concluded that it had not worked.

Team Plant Promoter

Team plant promoter were responsible for creating any constructs containing TSHH or Luc+ under the control of one of our 4 inducible promoters. They needed to isolate the promoters from Arabidopsis DNA, amplify them using PCR, and extract them, then put them into level 0 parts. Following that, they created level 1 constructs with these promoters, containing either TSHH or Luc+ as the CDS, and usually with the NOS terminator from Agrobacterium tumefaciens , but some of the final constructs somehow lacked these as shown on the composite parts page. Some of their results are shown below.

A photo of the DNA gel that showed the primers used to isolate our promoters from Arabidopsis had worked. We then extracted the DNA out of the gel, and used these for subsequent reactions. These are PDF1.2, PR2, , GST6, and WKRKY30 (not shown).

A photo of a gel showing successful level 0 WKRKY30 promoter constructs. Unfortunately, this is as far as WRKY30 got, as all subsequent level 1 reactions failed (gels not shown).

A DNA gel confirming three of our 8 level 1 constructs. These were the only Luc+ level 1 parts that Team Plant Promoter managed to successfully create, but when E. coli were transformed with this constructs, they continually failed to grow. This resulted in us running out of time in the lab, so sadly it was the furthest stage that our inducible promoter:Luc+:NosT constructs got to.

DNA gel showing that PDF1.2 and GST6 were both created with TSHH as the CDS. It was discovered that these lacked the NOS terminator when they were sequenced. PR2:TSHH was also created (not shown) and the three were infiltrated into N. benthamiana . The infiltrated leaves were then removed after inducition by the associated acid, and had a protein extract carried. This protein was then run on a gel, which can be seen below.

A protein gel showing the results of our protein extracts from leaves infiltrated with either GST:TSHH or PR:TSHH constructs. Following construct infiltration, they were then infiltrated with salicylic acid, left to express the gene, and cut off later. A protein extract was carried out on the leaves to assess whether or not our TSHH antagonist was present. By comparing with the negative control, we can see that these did not work. Perhaps with a longer incubation time, or with the NOS terminator present they would have.

A protein gel showing the results of infiltration PDF1.2:TSHH constructs, followed by infiltration of jasmonic acid. The lack of a band in the 'beads' samples, and the fact there is no band unique to infiltrated leaves when compared to the negative control suggests that this construct did not work. Perhaps with a longer incubation time, or with the NOS terminator present they would have.