Difference between revisions of "Part:BBa K4488012"

Latest revision as of 05:54, 10 October 2022

Fusion of free-use GFP with CBDcex (cellulose-binding domain) at the C-terminal end with a linker

The construct can be cloned into an expression vector such as pET28c in E.coli to produce a fusion protein of fuGFP with CBDcex. The fuGFP sequence is towards the N terminus of the protein with CBDcex (BBa_K4488023 ) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon.

Usage and Biology

Our project provided preliminary evidence that CBD and cellulose can be used to purify proteins by using fuGFP-linker-CBDcex.

Figure 1: Fluorescence measurements of filter paper binding test with fuGFP-linker-CBDs. 200 uL of TOP10-pUS250v3-fuGFP-CBD cell lysate was added to a 5 mm diameter paper filter disk and incubated for 1 h before washing with 200 uL NT buffer. Fluorescence measurements show that lysate with fusion proteins with the added linker exhibit more fluorescence than control cell lysate and makes the filter disks fluorescent.. fuGFP-linker-CBDcipA causes the greatest gain in fluorescence by paper filter disks.

The linker improved the solubility and fluorescence of the sequence. Below is the fluorescence assay depicting a higher reading compared to our previous construct BBa_K4488008 :

Figure 2: Fluorescence readings of fuGFP-linker-CBDs compared to fuGFP-CBDs in different E.coli plasmids. The uninduced and induced plasmids are also compared.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 163
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K4488012 short</partinfo>
-The construct can be cloned into an expression vector such as  pET28c in E.coli to produce a fusion protein of fuGFP with CBDcex. The fuGFP sequence is towards the N terminus of the protein with CBDcex (BBa_K1321342) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon.
+The construct can be cloned into an expression vector such as  pET28c in E.coli to produce a fusion protein of fuGFP with CBDcex. The fuGFP sequence is towards the N terminus of the protein with CBDcex (BBa_K4488023 ) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon.
-Below is the fluorescence assay depicting a higher reading compared to our previous construct BBa_K4488008 :
-[TBA]
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+Our project provided preliminary evidence that CBD and cellulose can be used to purify proteins by using fuGFP-linker-CBDcex.
+[[File: fuGFP-linker-CBD washes.png|centre|thumb|700px|Figure 1: Fluorescence measurements of filter paper binding test with fuGFP-linker-CBDs. 200 uL of TOP10-pUS250v3-fuGFP-CBD cell lysate was added to a 5 mm diameter paper filter disk and incubated for 1 h before washing with 200 uL NT buffer. Fluorescence measurements show that lysate with fusion proteins with the added linker exhibit more fluorescence than control cell lysate and makes the filter disks fluorescent.. fuGFP-linker-CBDcipA causes the greatest gain in fluorescence by paper filter disks.]]
+The linker improved the solubility and fluorescence of the sequence. Below is the fluorescence assay depicting a higher reading compared to our previous construct BBa_K4488008 :
+[[File:CBD Engineering Cycle.png|centre|thumb|700px|Figure 2: Fluorescence readings of fuGFP-linker-CBDs compared to fuGFP-CBDs in different E.coli plasmids. The uninduced and induced plasmids are also compared.]]
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