Difference between revisions of "Part:BBa K4159012"

Latest revision as of 17:15, 1 October 2022

GFP binding DARPin for expression

Description: This is the GFP DARPin in a composite part for expression of the DARPin. The promoter used in the construct is T7(Hansen et al., 2017), after that RBS sequence and then two different tags for downstream processing. The His-tag is mainly for purification and the avi-tag is for binding assays. After that the actual DARPin sequence, and then an additional His-tag and stop codon. The whole part has been ordered as one from IDT in our project and the needed restriction sites can be added on easily with overhangs in PCR.

Characterization: Amplification of the part that will be integrated into the pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS plasmid. The plasmid and part were digested with Xbal and NcoI. The part was ligated and transformed into BL21 expression cells. The cells were induced with IPTG, and extracted using sonication. The protein was then purified with Ni-NTA spin columns. In figure 1. We show that we successfully expressed the GFP DARPin at the correct size ~17kDa. The protein was mostly in the pellet, which means that further optimizations are needed to get the protein into the supernatant for Ni-NTA purification, or testing other extraction methods for extracting the DARPins from the pellet directly.

SDS-page gel picture of the expressed GFP-binding DARPin

References: Hansen S, Stüber JC, Ernst P, Koch A, Bojar D, Batyuk A, Plückthun A. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity. Sci Rep. 2017 Nov 24;7(1):16292. doi: 10.1038/s41598-017-15711-z. PMID: 29176615; PMCID: PMC5701241.

https://www.ncbi.nlm.nih.gov/protein/5MA6_B

HIS-tag reference pET Biotin His6 GFP LIC cloning vector (H6-msfGFP) was a gift from Scott Gradia (Addgene plasmid # 29725 ; http://n2t.net/addgene:29725 ; RRID:Addgene_29725)

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 21
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 21
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 21
1000
COMPATIBLE WITH RFC[1000]

@@ Line 4: / Line 4: @@
 Description: This is the GFP DARPin in a composite part for expression of the DARPin. The promoter used in the construct is T7(Hansen et al., 2017), after that RBS sequence and then two different tags for downstream processing. The His-tag is mainly for purification and the avi-tag is for binding assays. After that the actual DARPin sequence, and then an additional His-tag and stop codon. The whole part has been ordered as one from IDT in our project and the needed restriction sites can be added on easily with overhangs in PCR.
 Characterization:
-Amplification of the part that will be integrated into the pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS plasmid. plasmid and part was digested with Xbal and NcoI. The part was ligated and transformed into BL21 expression cells. The cells were induced with IPTG, and extracted using sonication. The protein was then purified with Ni-NTA spin columns.
+Amplification of the part that will be integrated into the pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS plasmid. The plasmid and part were digested with Xbal and NcoI. The part was ligated and transformed into BL21 expression cells. The cells were induced with IPTG, and extracted using sonication. The protein was then purified with Ni-NTA spin columns. In figure 1. We show that we successfully expressed the GFP DARPin at the correct size ~17kDa. The protein was mostly in the pellet, which means that further optimizations are needed to get the protein into the supernatant for Ni-NTA purification, or testing other extraction methods for extracting the DARPins from the pellet directly.
+[[File:BBa K415012 1.png|200px|thumb|left|SDS-page gel picture of the expressed GFP-binding DARPin]]
+References:
+Hansen S, Stüber JC, Ernst P, Koch A, Bojar D, Batyuk A, Plückthun A. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity. Sci Rep. 2017 Nov 24;7(1):16292. doi: 10.1038/s41598-017-15711-z. PMID: 29176615; PMCID: PMC5701241.
+ https://www.ncbi.nlm.nih.gov/protein/5MA6_B
+HIS-tag reference
+pET Biotin His6 GFP LIC cloning vector (H6-msfGFP) was a gift from Scott Gradia (Addgene plasmid # 29725 ; http://n2t.net/addgene:29725 ; RRID:Addgene_29725)