Team TSH consisted of Emily, Helen and Sarah. The objective of this team was to express the thyroid stimulating hormone antagonist in two different forms.
These were with 'His tags' and without. Both of these constructs can be found on the basic parts page. This team was responsible for creating the level one constructs with the 35S or LexA promoter containing the TSH or TSHH CDS.
The level zero constructs were put together using the type IIS restriction enzyme BsmB1 at 55 degrees C (Not 37!). These level 0 parts were then combined to create the level 1 constructs mentioned above using Bsa1 which (thankfully) does cut at 37 degrees. These constructs were used to transform Agrobacterium , which was infiltrated into our Nicotiana benthamiana plants. In adddition we worked with Team Plant_P to express TSHH using the plant promotors that they have generated as level 0 parts.
At the end of the project although we had not optimised the TSH protein expression, we know our tobacco transformations worked as demonstrated by the luciferase reporter gene work performed by team luciferase so we are hopeful that this will occur in the future.
Therefore we submited these TSH level 0 parts to the registry after confirming them by sequencing! Finally although we ran out of time in the lab to optimse the expression assays, Sarah will be continuing this work in a future research project.