Part:BBa_K3077100
Expression of lip8 under control of lac promoter
This part BBa_K3077100, is the composite part of the cytoplasmic esterase encoded as lip8 in Pseudomonas aeruginosa, which is registered as BBa_K3077101 . The genomic sequence of the lip8 gene is referenced from [1]. The lip8 gene is designed to be the composite part BBa_K3077100 by adding lac promoter BBa_R0010, strong RBS BBa_B0034, 10x-Histidine tag BBa_K844000 and double terminator BBa_B0015.
Basic Parts
- R0010 is a conditional promoter that promotes transcription in the absence of LacI protein and CAP protein, which can be maintained by the presence of IPTG and low glucose level. This can prevent overexpression of the cell and keep it healthy.
- B0034, a strong RBS, to achieve a higher expression level.
- K844000, a 10X His-tag, to allow protein purification, is fused at the N terminus of the lipase gene.
- B0015 is a double terminator combining B0010 and B0012, and is one of the most commonly used and reliable terminator in the iGEM catalog.
Coding Gene
The gene BBa_K3077101 is a cytoplasmic esterase encoded as lip8 in Pseudomonas aeruginosa. Lip8 is an esterase that catalyzes both the hydrolysis and synthesis of ester bonds. It is also a biocatalyst for interesterification, alcoholysis, acidolysis, and aminolysis.[2] It showed higher activities against short-chain fatty acid methyl esters compared with activities against various triacylglycerols and long-chain fatty acid methyl esters: Among triacylglycerols with different fatty acids, Lip8 was found to have the highest activity against triacetin (C2). While methyl acetate (C2), methyl propionate (C3), and methyl butyrate (C4) are the substrates with higher hydrolytic activity among different fatty acid methyl esters. Among four Natural oils, Lip8 showed highest activity against tung oil, whose principal fatty acid component is eleostearic acid (C18: 3)[2].
This plasmid containing composite part BBa_K3077100 is ordered and synthesized by IDT. The ordered plasmid is transformed in E.coli and is verified through colony PCR with prefix forward and suffix reverse. We tested its catalytic activity as an esterase through the lipid hydrolysis assay.
Assay
We carried out two assays to test the presence and effectiveness of the transformed E.coli containing BBa_K3077100. Firstly, we verified the presence of the DNA by colony PCR followed by gel electrophoresis. Secondly, we tested the catalytic activity on Tributyrin Agar of the bacteria.
Assay 1: DNA verification
Referring to the DNA ladder, clone 1,2,3,5,6 collectively showed bands at about 1.6 kB, which fits the sequence length of the composite part BBa_K3077100 of 1580 bp, thus verified the presence of the correct insert. On the other hand, clone 4 failed. The E.coli of the correct clones are then amplified and used in the assay below.
Assay 2: Lipid hydrolysis assay
We tested the catalytic activity of the transformed composite part BBa_K3077100 with 91010 Tributyrin agar. E.coli transformed with the composite part plasmid was spread on a Tributyrin agar plate. The cells were incubated for 1 day.
Observations:
Of the 5 sets of data, only 2 clones showed the growth of clear zone underneath the E.coli, but overall there is no clear positive results.
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Clones that showed less lipid droplets underneath the E.coli
This can be explained by the esterase(Lip8) being cytoplasmic, which makes it inaccessible to its substrate in the external environment unless the substrate passes through the outer membrane, or the esterase leaks out of the cell in a significant amount. Therefore, we plan to carry out the Lipid hydrolysis assay again after cell lysis to test its cytoplasmic catalytic activity. Yet, we can’t do as planned due to insufficient time and it will be continued in our next year project.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 520
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 738
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