Part:BBa_K5088150

Plant GFP with IV2 intron

View the results of this basic part.
Check out the corresponding measurement construct by clicking here.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Background

To improve the sensitivity and reliability of our part characterization, we transitioned from using the original Aequorea victoria GFP (avGFP) - which was provided in the iGEM distribution kit - to an enhanced variant, eGFP. The avGFP, isolated in 1962, exhibited lower brightness, which likely contributed to the suboptimal signal observed in our experiments. In contrast, eGFP is a modern, brighter fluorescent protein that significantly enhances assay performance by providing a stronger and more detectable fluorescence signal.

Additionally, to mitigate the risk of false-positive fluorescence arising from unintended transcription in Agrobacterium, we incorporated the potato ST-LS1 intron (IV-2 intron) into the eGFP coding sequence. This intron effectively prevents Agrobacterium from expressing the eGFP, as plant promoters in Agrobacterium can inadvertently initiate transcription, leading to misleading fluorescence signals. The insertion of the IV-2 intron ensures that eGFP expression is restricted to plant tissues, thereby enhancing the specificity and accuracy of our fluorescence measurements.

Parts we have tested using this reporter

Part Identifier	Part Type	Nickname	Part Description
BBa_K5088001	Promoter + 5'UTR	P_RPL28	Large subunit ribosomal protein L28e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088006	Promoter + 5'UTR	P_FKBP4_5	FK506-binding protein 4/5 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088007	Promoter + 5'UTR	P_CLTC	Clathrin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088008	Promoter + 5'UTR	P_RPL31	Large subunit ribosomal protein L31e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088012	Promoter + 5'UTR	P_Tubulin	Tubulin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088013	Promoter + 5'UTR	P_EIF5A	Translation initiation factor 5A - Promoter+5'UTR from T. kok-saghyz
BBa_K5088102	3'UTR	T_PTI1	Protein tyrosine kinase - 3'UTR from T. kok-saghyz
BBa_K5088103	3'UTR	T_RPL28	Large subunit ribosomal protein L28e - 3'UTR from T. kok-saghyz
BBa_K5088104	3'UTR	T_EPS15	Epidermal growth factor receptor substrate 15 - 3'UTR from T. kok-saghyz
BBa_K5088105	3'UTR	T_GSK3B	Glycogen synthase kinase 3 - 3'UTR from T. kok-saghyz
BBa_K5088106	3'UTR	T_MGRN1	E3 ubiquitin-protein ligase - 3'UTR from T. kok-saghyz
BBa_K5088107	3'UTR	T_RPL35A	Large subunit ribosomal protein L35Ae - 3'UTR from T. kok-saghyz
BBa_K5088108	3'UTR	T_betB	Betaine-aldehyde dehydrogenase - 3'UTR from T. kok-saghyz
BBa_K5088109	3'UTR	T_pgm	Phosphoglucomutase - 3'UTR from T. kok-saghyz
BBa_K5088110	3'UTR	T_ATP-synt	ATPase subunit gamma - 3'UTR from T. kok-saghyz
BBa_K5088111	3'UTR	T_EIF3B	Translation initiation factor 3 subunit B - 3'UTR from T. kok-saghyz
BBa_K5088112	3'UTR	T_RPL31	Large subunit ribosomal protein L31e - 3'UTR from T. kok-saghyz
BBa_K5088113	3'UTR	T_TM9SF2_4	Transmembrane 9 superfamily member 2/4 - 3'UTR from T. kok-saghyz
BBa_K5088114	3'UTR	T_CUL1	Cullin - 3'UTR from T. kok-saghyz
BBa_K5088115	3'UTR	T_PSMB6	20S proteasome subunit beta 1 - 3'UTR from T. kok-saghyz
BBa_K5088116	3'UTR	T_RPSA	Small subunit ribosomal protein SAe - 3'UTR from T. kok-saghyz
BBa_K5088117	3'UTR	T_VPS4	Vacuolar protein-sorting-associated protein 4 - 3'UTR from T. kok-saghyz
BBa_K5088118	3'UTR	T_EIF2S3	Translation initiation factor 2 subunit 3 - 3'UTR from T. kok-saghyz

Table 1: List of functioning T. kok-saghyz endogenous regulatory elements we've characterized in our project

Results

Leaf Infiltration

Taraxacum

Figure : Fluorescence bino images (BF= Brightfield, AF= Autofluorescence Ex: Em:, mCherry Ex: Em: , eGFP Ex: Em: ) A)Transgenic TKS leaves, infiltrated with Tarakate - Consensus measurement construct [BBa_K5088677]. B) Transgenic TO leaves, infiltrated with Tarakate - Consensus measurement construct [BBa_K5088677]. C) WT TKS, showing no mCherry and eGFP expression.

N. benthamiana

Figure : Comparison between Tarakate - Test construct GFP [BBa_K5088675] and Tarakate - Consensus measurement construct [BBa_K5088677]. The control represents non infiltrated leaf disk of N. benthamiana, K599 show leaf disks infiltrated with A. rhizogenes K599 and A. tumefaciens GV3101 P19 as additional negative controls. A) avGFP expression [Ex: 408nm ± 6nm; Em: 515nm ± 6nm] B) mCherry expression [Ex: 570nm ± 6nm; Em: 610nm ± 6nm] C) eGFP expression [Ex: 472nm ± 6nm; Em: 515nm ± 6nm].

Figure : Measurement of Promoter + 5'UTR parts in leaf disk assay of N. benthamiana. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Figure : Measurement of 3'UTR parts in leaf disk assay of N. benthamiana. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Protoplast

Figure : Fluorescense microscopy images (Nikon Ti, camera: Andor Zyla VSC-01427, objective: Plan APO VC 20x CIC N2, mCherry: Ex 575 nm, Em 647 nm, eGFP: Ex 470 nm, Em 525 nm). A) WT TKS protoplasts, showing no mCherry and eGFP expression. B) Transgenic TKS protoplasts, transfected with BBa_K5088614, showing mCherry and eGFP expression 17h post transfection.

Figure : Measurement of 3'UTR parts in protoplasts of T. kok-saghyz. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Figure : Measurement of 3'UTR parts in protoplasts of T. officinale. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Cut-Dip-Budding

Figure : Microscopy image of Cut dip budding transformed Taraxacum roots. Each subfigure is subdivided into four panels, where BF stands for brightfield and AF for autofluorescence. Shown below is the according mCherry and eGFP signal of the same root. A) Negative control B) Tarakate - Consensus measurement construct [BBa_K5088677] in T. kok-saghyz C) Tarakate - Consensus measurement construct [BBa_K5088677] in T. officinale

Figure :Microscopy image of Cut dip budding transformed Taraxacum roots. Each subfigure is subdivided into four panels, where BF stands for brightfield and AF for autofluorescence. Shown below is the according mCherry and eGFP signal of the same root. A) Negative control of T. kok-saghyz B) Measurement construct - Tubulin promoter+5'UTR from T. kok-saghyz [BBa_K5088512] tested in T. kok-saghyz.