Difference between revisions of "Part:BBa K4340600"

(→‎Experiment 2: Western Blot and Real-time PCR)
 
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[[File:hormone test 1.png|400px|thumb|center|Figure 1. The seed germination percentage of soybeans in seven days with all factors.]]
 
[[File:hormone test 1.png|400px|thumb|center|Figure 1. The seed germination percentage of soybeans in seven days with all factors.]]
 
[[File:hormone test t2r4.png|400px|thumb|center|Figure 2. The seed germination percentage of soybeans in seven days with NSP4-T2R4.]]
 
[[File:hormone test t2r4.png|400px|thumb|center|Figure 2. The seed germination percentage of soybeans in seven days with NSP4-T2R4.]]
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[[File:Hbps soybeans new.jpeg|600px|thumb|center|Photo 1. Germination of soybeans with or without added ABA, and either with PYL8, NSP4-T2R4 or empty pET11a vector transformed E. coli.]]
  
 
To sum up, the efficiency of the plasmids ranked is PYL8> NSP4-T2R4 (slightly higher than) pET11a.
 
To sum up, the efficiency of the plasmids ranked is PYL8> NSP4-T2R4 (slightly higher than) pET11a.
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==Experiment 2: Western Blot and Real-time PCR==
 
==Experiment 2: Western Blot and Real-time PCR==
  
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[[File:PYL8 T2R4 westernblot.jpg|400px|thumb|center|Figure 1. The protein expression of NSP4-T2R4 and PYL8]]
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[[File:PYL8 T2R4 westernblot.jpeg|600px|thumb|center|Figure 1. The protein expression of NSP4-T2R4 and PYL8.]]
  
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[[File:T2R4 pcr.jpeg|400px|thumb|center|Figure 2: The real-time PCR result of NSP4-T2R4 compared with BL21 E.coli strain.]]
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Figure 2: The real-time PCR result of NSP4-T2R4 compared with BL21 E.coli strain.
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We have conducted a western blot experiment to validate the quality of protein expression of NSP4-T2R4. In the experiment,  there is a relatively more unclear band of NSP4-T2R4. (Figure 1) Therefore, further experiments would be needed to confirm whether no protein expression is the reason why T2R4 cannot inhibit ABA.
 
We have conducted a western blot experiment to validate the quality of protein expression of NSP4-T2R4. In the experiment,  there is a relatively more unclear band of NSP4-T2R4. (Figure 1) Therefore, further experiments would be needed to confirm whether no protein expression is the reason why T2R4 cannot inhibit ABA.
  
 
However, in the real-time quantitative PCR test, we found the RNA expression of NSP4-T2R4 genes, indicating there could be some protein expression problem with T2R4, but not RNA. (Figure 2)
 
However, in the real-time quantitative PCR test, we found the RNA expression of NSP4-T2R4 genes, indicating there could be some protein expression problem with T2R4, but not RNA. (Figure 2)
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Latest revision as of 22:58, 11 October 2022


T2R4 with NSP4 peptides

T2R4 is one of the ABA binding proteins and adding NSP4, the secretion peptide is helpful to secrete more proteins. We tested whether the hormone-binding domain can inhibit and attenuate the effect of ABA (plant hormone that inhibits germination and plant growth) to plant germination. The pET11a and PYL8 E.coli culture groups, and a group with ABA treatment and PYL8 E. coli culture had a higher germinated percentage than the group without ABA (blank). This indicated that the PYL8 had a positive effect on the seed germination stage both with ABA and without ABA. (Figure 1)

NSP4-T2R4, however, had the same function as the pET11a vector control, (Figure 2) which means that NSP4-T2R4 might not have an outstanding effect on increasing the speed of germination of soybeans.

Experiment 1: Hormone-treated germination test

Figure 1. The seed germination percentage of soybeans in seven days with all factors.
Figure 2. The seed germination percentage of soybeans in seven days with NSP4-T2R4.
Photo 1. Germination of soybeans with or without added ABA, and either with PYL8, NSP4-T2R4 or empty pET11a vector transformed E. coli.

To sum up, the efficiency of the plasmids ranked is PYL8> NSP4-T2R4 (slightly higher than) pET11a.

Experiment 2: Western Blot and Real-time PCR

Figure 1. The protein expression of NSP4-T2R4 and PYL8.
Figure 2: The real-time PCR result of NSP4-T2R4 compared with BL21 E.coli strain.

We have conducted a western blot experiment to validate the quality of protein expression of NSP4-T2R4. In the experiment, there is a relatively more unclear band of NSP4-T2R4. (Figure 1) Therefore, further experiments would be needed to confirm whether no protein expression is the reason why T2R4 cannot inhibit ABA.

However, in the real-time quantitative PCR test, we found the RNA expression of NSP4-T2R4 genes, indicating there could be some protein expression problem with T2R4, but not RNA. (Figure 2)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 346
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 48
  • 1000
    COMPATIBLE WITH RFC[1000]