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[http://2018.igem.org/Team:Cardiff_Wales Cardiff Wales 2018]

Testing Phytobrick Terminators
Introduction

The iGEM [http://2016.igem.org/Resources/Plant_Synthetic_Biology/PhytoBricks Phytobrick registry] contains three terminator phytobricks for plant expression.

> The NOS terminator > The G7 terminator > The 3U+Ter-CaMV35S terminator

The registry contains no information about expression of genes from clones that include any of these terminator sequences. Therefore we aimed to test each of these terminator in the tobacco leave expression system.

Methods

These terminator sequences are level 0 golden gate clones. These parts were added to the pGB-A2 level 1 plasmid in a reaction with the constitutive 35S promotor, the transcriptional enhancer OTMV and the GUS reporter gene. This reporter gene was submitted to the registry by the [http://2018.igem.org/Team:Cardiff_Wales Cardiff_Wales 2018 team].

Therefore these composite level 1 clones are as follows: > pGB-35S-OTMV-GUS-Nos > pGB-35S-OTMV-GUS-G7 > pGB-35S-OTMV-GUS-3U+Ter-CaMV35S

These level 1 composite parts specifically for plant expression cannot be submitted to the iGEM registry as they are not contained within the pSB1C series of plasmids.

These clones were introduced into agrobacterium strain GV3101 (see [http://2018.igem.org/Team:Cardiff_Wales/Protocols Cardiff_Wales Wiki for the protocol]).

Subsequently transformed agrobacteria were infiltrated into leaves of Nicotiana benthamiana. After 2days the transformed leaves were heat shocked at 37C for 30minutes to improve protein expression. After a further day leaf discs were removed from infiltrated leaves and stained overnight with GUS staining solution. GUS staining solution was removed and the leaves were cleared in 100% ethanol.

For all protocols see [http://2018.igem.org/Team:Cardiff_Wales/Protocols Cardiff_Wales protocol page on our Wiki].
Results

Four leaves were infiltrated with each experimental construct as well as with an empty level 1 plasmid control. Multiple leaves were used to mitigate the known variation in expression that can occur between leaves following difference infiltrations.

This experiment was repeated twice and shown in Figure 1.

Figure 1) The GUS staining assay to quantify the Nopaline synthase terminator, 35S terminator, and G7 terminator. This suggests that the G7 terminator is the strongest.

Figure 1 shows that there is consistent expression in discs taken from leaves infiltrated with the same construct, demonstrating that the protocol for infiltration and subsequent analysis of gene expression was successful.

Across both experiments leaves expressing 35S-OTMV-GUS-G7 have consistently higher expression than leaves expressing the other two constructs. Over the course of the two experiments it appears that leaves expressing 35S-OTMV-GUS-Nos have less consistent and potentially lower expression than leaves expressing the other two constructs.

Conclusions

The iGEM Phytobrick registry contains no information about the efficacy of different terminator elements for plant expression. Therefore in these experiments the [http://2018.igem.org/Team:Cardiff_Wales Cardiff_Wales iGEM team] generated a set of three clones using a newly generated GUS reporter gene in which expression was controlled by different terminator elements.

Our experiments found that use of each terminator was effective for control of gene expression but that the G7 terminator might show improved expression. These experiments indicate that different terminator elements should be tested in future experiments if researchers have concerns about maximising gene expression.

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