Part:BBa_K3347005
Expression block for Dehalogenase type 1.variant 2.non-tagged (DeHa1.2.gene; BBA_K3347004) for use in BL21-DE3 (pLysS) E. coli. This part's construction was inspired primarily from the standard pET-28 vector (T7 polymerase, RBS, and terminator) and adding DeHa1.2.gene as a coding sequence. This sequence allows the expression of Dehalogenase type I from Delftia acidovorans upon induction from IPTG using the T7 polymerase gene in the bacterial chromosome under expression control of LacUV5, an improved lac promoter.
New to 2021
USAFA iGEM 2021 Contribution:
The 2020 USAFA iGEM team left the BBa_K3347005 part as a theoretically functional dehalogenase enzyme. The 2021 USAFA iGEM expanded the work of the 2020 team and successfully expressed the BBa_K3347005 dehalogenase in E. coli and purified an active enzyme. Initial data from fluorine NMR suggests this purified enzyme cleaves off the terminal trifluoromethyl group of PFOA. This new data validates the BBa_K3347005 part is a viable enzyme.
Figure 1: DeHa 1 was expressed in BL21 and then purified. Performing protein densitometry analysis on the diluted, purified enzymes revealed the concentration of purified DeHa 1 was 16.81 mg/mL
Naming notation for Dehalogenase genes and expression blocks
Plasmid notation:
Represented by pXX_DeHa
Where XX can represent:
C: pSB1C3
A: pSB1A3
K: pSB1K3
KB: pSRKBB
Dehalogenase gene & Expression Block Notation
Represented by DeHaX.Y.Z
Where,
X is the gene type (1 or 2 ; referencing the two dehalogenase genes in D. acidovorans)
Y is the variant (native variant = 1, E. coli codon optimized = 2, etc.)
Z shows the tags/modifications on the protein (0 = no modifications, HisC = 6xHis tag on carboxyl terminal, HisN = 6xHis tag on amine terminal, gene, etc.)
Note: If Z is replaced by the 'gene' modifier, the Dehalogenase gene sequence is being referenced. In all other cases, the fully assembled expression block is referenced. Example: DeHa1.1.gene references BBa_K3347000 while DeHa1.1.0 references BBa_K3347001.
Example notations
pKB_DeHa2.2.HisN: Dehalogenase type II, variant 2 (E. coli optimized) with a N terminal 6xHis tag all inside the pSRKBB vector
pC_DeHa1.1.0: Dehalogenase type I, variant 1 with no further tags or modifications all inside the pSB1C3 vector
This expression system is used for large protein expression of DeHa1.2 using the vector pSRKBB (pKB_DeHa1.2.0) inside the chassis BL21-DE3 (pLysS) E. coli
Sequence and Features
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