Difference between revisions of "Part:BBa K4040026"
| Line 15: | Line 15: | ||
===Process of experiment=== | ===Process of experiment=== | ||
We used mCherry (<partinfo>BBa_J06504</partinfo>) as the reporter and connected it to the 8×PIR minCMV promoter (<partinfo>BBa_K3132101</partinfo>). The cells were co-transfected with GEMS(BBa_K4040025, BBa_K4040026, BBa_K4040027, BBa_K4040028, BBa_K4040029), reporter plasmid, and PIP-Elk1 respectively. Then they were added with the IL-6*Suntag proteins at 24h post-transfection. Eighteen hours later, the mCherry fluorescence intensity was measured. The fluorescence intensity when IL-6 concentration is 100ng/ mL and 0ng/ mL is shown in Fig.2. | We used mCherry (<partinfo>BBa_J06504</partinfo>) as the reporter and connected it to the 8×PIR minCMV promoter (<partinfo>BBa_K3132101</partinfo>). The cells were co-transfected with GEMS(BBa_K4040025, BBa_K4040026, BBa_K4040027, BBa_K4040028, BBa_K4040029), reporter plasmid, and PIP-Elk1 respectively. Then they were added with the IL-6*Suntag proteins at 24h post-transfection. Eighteen hours later, the mCherry fluorescence intensity was measured. The fluorescence intensity when IL-6 concentration is 100ng/ mL and 0ng/ mL is shown in Fig.2. | ||
| − | [[File:T-- | + | [[File:T--NMU_China--gems.png|500px|thumb|center| <b>Figure. 2</b> mCherry fluorescence intensity when the IL-6 concentration is 0ng/ml and 100ng/ml respectively.***P<0.001, *P<0.05 ]] |
The experimental results showed that when the concentration of IL-6 was 0, the fluorescence signal intensity gradually decreased with the increase of the inserted glutamate. But when the concentration of IL-6 was 100ng/ml, the fluorescence intensity of GEMS (<partinfo>BBa_K4040029</partinfo>) inserted with four glutamate sequences is significantly lower than that of the original GEMS and the GEMS inserted with 1-3 glutamate sequences. For GEMS (<partinfo>BBa_K4040028</partinfo>) with an increase of 3 glutamates, the signal conduction activity can be guaranteed while the background activity is low. So we consider GEMS inserted with 3 glutamate sequences to be the best improvement part. | The experimental results showed that when the concentration of IL-6 was 0, the fluorescence signal intensity gradually decreased with the increase of the inserted glutamate. But when the concentration of IL-6 was 100ng/ml, the fluorescence intensity of GEMS (<partinfo>BBa_K4040029</partinfo>) inserted with four glutamate sequences is significantly lower than that of the original GEMS and the GEMS inserted with 1-3 glutamate sequences. For GEMS (<partinfo>BBa_K4040028</partinfo>) with an increase of 3 glutamates, the signal conduction activity can be guaranteed while the background activity is low. So we consider GEMS inserted with 3 glutamate sequences to be the best improvement part. | ||
Revision as of 10:55, 11 October 2021
GEMS with one Alanine added
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2447
Illegal XhoI site found at 2736 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1189
Illegal NgoMIV site found at 1300
Illegal NgoMIV site found at 1312
Illegal NgoMIV site found at 1744 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4
Illegal SapI.rc site found at 1569
Illegal SapI.rc site found at 1584
Illegal SapI.rc site found at 1920
Background of the improvement parts
This part is a mutant of GEMS designed by team SMMU in 2019. GEMS(BBa_K3032016) is a dimer of two subunits of which the scaffold is derived from EpoR (erythropoietin receptor). GEMS can be combined with a wide range of affinity domains as well as different intracellular domains for rerouting signaling to activate distinct endogenous signaling pathways [1]. In the previous paper, the authors rewired the receptors to four independent pathways (Figure. 1b), and these receptors could respond to various molecules of different molecular weights. BBa_K3032016 is a MAPK-dependent GEMS that consists of an anti-Suntag scFV and a FGFR1 intracellular domain. Therefore, this receptor is designed to specifically respond to proteins fused with SunTag and to activate MAPK pathway.
Design
In the experiment of exploring the influence of the amount of SunTag on protein activity by team SMMU in 2019, they used mCherry (BBa_J06504) as the reporter and connected it to the 8×PIR minCMVpromoter (BBa_K3132101). The cells were co-transfected with GEMS, reporter plasmid, and PIP-Elk1, and were added with the IL-Suntag proteins 24h post-transfection. Eighteen hours later, the mCherry fluorescence intensity was measured. Intriguingly, among the three ligands, only 6×SunTag showed significant activating effect (Figure. 1a). The receptor showed around 3.2-fold change at the concentration of 100ng/ml compared with negative control. However, weak fluorescence could be observed in the negative control, suggesting that this receptor possessed a low background activity (Figure. 1b).
This year we hope to change the structure of the GEMS to reduce this base activity and design experiments accordingly. We learned from the reference that the GEMS receptor would rotate when adding different amounts of glutamate between the scFV and the intracellular domains of FGFR1, causing the receptor to form both ON and OFF states, which alters the activity of the receptor transduction signal. Inspired by this phenomenon, we added 0, 1, 2, 3 and 4 alanine sequences between the anti-Suntag scFV and the internal domains of FGFR1 cells respectively on the basis of GEMS (BBa_K3132016). We then verified the background expression of this receptor when the concentration of IL-6 was 0.
Process of experiment
We used mCherry (BBa_J06504) as the reporter and connected it to the 8×PIR minCMV promoter (BBa_K3132101). The cells were co-transfected with GEMS(BBa_K4040025, BBa_K4040026, BBa_K4040027, BBa_K4040028, BBa_K4040029), reporter plasmid, and PIP-Elk1 respectively. Then they were added with the IL-6*Suntag proteins at 24h post-transfection. Eighteen hours later, the mCherry fluorescence intensity was measured. The fluorescence intensity when IL-6 concentration is 100ng/ mL and 0ng/ mL is shown in Fig.2.
The experimental results showed that when the concentration of IL-6 was 0, the fluorescence signal intensity gradually decreased with the increase of the inserted glutamate. But when the concentration of IL-6 was 100ng/ml, the fluorescence intensity of GEMS (BBa_K4040029) inserted with four glutamate sequences is significantly lower than that of the original GEMS and the GEMS inserted with 1-3 glutamate sequences. For GEMS (BBa_K4040028) with an increase of 3 glutamates, the signal conduction activity can be guaranteed while the background activity is low. So we consider GEMS inserted with 3 glutamate sequences to be the best improvement part.
Meaning of improvement
Improved GEMS (BBa_K4040028) can reduce spontaneous activation while preserving receptor activity, so this modification can improve specificity and enhance the function of GEMS receptor without reducing receptor sensitivity.
References
[1] Scheller, L., et al., Generalized extracellular molecule sensor platform for programming cellular behavior. Nat Chem Biol, 2018. 14(7): p. 723-729.