Team:Tokyo-NoKoGen/experiments/Lab note

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July 26 ~August 1 [solubilization] Culturing of pseudomonas aeruginosa
TA cloning of RhlA and RhlB genes from pseudomonas aeruginosa

[Tank] Purchased Citrobacter freundii (Braak1928) Werkman and Gillen 1932

August 2 - August 8 [solubilization] We carried out sequences analysis of RhlA and RhlB, but we could not confirm the sequence.

[Tank] Amplification of the genes (pduAB, pduJK, pduN, pduU) respectively by PCR from Citrobacter freundii (Braak1928)

August 9 - August 15 [Tank] The PCR product (pduAB, pduJK, pduN, pduU) were inserted into pSB vector.
DH5 were transformed by the constructed plasmids.
Colony PCR was carried out to confirm the constructions.
Extraction of plasmids.
Sequence analysis of cloned genes

  • ・Analyzed the sequence of last-week extracted plasmids
    • ・pduAB, pduN and pduU were almost same as the sequence registered in EMBL Nuceotide Sequence Database (accession number AM498294) ・pduJK had a little different sequence from the sequence of AM498294.We thought that the difference might be due to the difference between species; C.freundii and registered one.

August 16 - August 22 [solubilization] TA cloning of RhlA and RhlB genes from pseudomonas aeruginosa again
Subcloning of RhlA and RhlB
Insertion of RhlA and RhlB genes into pSB vector

[Tank] pduAB and pduJK genes have Pst1 sites. So we removed the Pst1 sites in pduAB and pduJK by overlap PCR

August 23 - August 29 [Tank] Confirmation of modification of Pst1 site in pduAB and pduJK by sequence analysis
Amplification of pduAB, pduJK, pduN, pduU genes using primers which attached RBS(B0034) to each gene

August 30 - September 5 [solubilization] Construction of promoter-RBS-RhlA-term and promoter-RBS-RhlB-term parts using megaprimer made by overlap PCR.
The PCR product (promoter-RBS-RhlA-term and promoter-RBS-RhlB-term) was inserted into pSB vector
Transformation of E. coli DH5α by the plasmid (We did not get white colony)

[Tank] Construction of pduABJK by 3A assembly
Construction of pduU-terminator

[Phototaxis] Overlap extension PCR & Sequence analysis
failed→SRll-Htrll-Tar
succeed→Htrll (pSB1C3-Htrll sequence was comfirmed)

[Photocontrol] Preparation of plasmid
OmpR promoter (BBa_R0082)
RBS-GFP-Term
Construction of OmpR-promoter-RBS-GFP-Term by 3A assembly
Sequence analysis of OmpR-promoter-RBS-GFP-Term (we could not confirm the sequence.)

[Aggregation] TA-cloning of Antigen 43
pGEMT-Antigen 43(Wildtype) was constructed

September 6 - September 12 [Tank] Construction of pduNU-terminator

[Phototaxis] Construct of pSB1C3-SRll-Htrll-Tarm

[Photocontrol] Construction of OmpR-promoter-RBS-GFP-Term again.
Sequence analysis of OmpR-promoter-RBS-GFP-Term (we confirmed the sequence!!)

[Aggregation] Subcloning of Antigen 43(Wildtype) pSB1C3-Antigen 43(Wild type) was constructed.
Removal of the 6 PstI sites in Antigen 43(Wild type) by PCR.

September 13 - September 19 [Tank] Construction of pduABJKNU-terminator

[Phototaxis] Sequence analysis
pSB1C3-Tar, pSB1C3-SRll and pSB1C3-SRll-Htrll-Tar sequence were comfirmed

[Photocontrol] Preparation of the plasmid
RBS-ho1-RBS-PcyA-RBS-GlrN-Trem
Construct of RBS-ho1-RBS-PcyA-RBS-GlrN-Trem- OmpR promoter-RBS-GFP-Trem

[Aggregation] PstI sites modified-Antigen 43 gene was inserted into pSB1C3.
Construct of pSB1K3-RBS-Antigen 43

September 20 - September 26 [Phototaxis] Construction of pSB1C3-RBS-SRll-Htrll-Tar by 3A assembly (sequence couldn’t be comfirmed)

[Photocontrol] Construct of RBS-ho1-RBS-PcyA-RBS-GlrN-Trem- OmpR promoter-RBS-GFP-Trem again
Sequence analysis of the construct (we confirmed the sequence!!)

[Aggregation] Construct of pSB1K3-RBS-Antigen 43 Double terminator

September 27 - October 3 [Tank] Construction of pduP-GFPuv-terminator

[Phototaxis] Construction of pSB1C3-RBS-SRll-Htrll-Tar by 3A assembly (sequence couldn’t be comfirmed) twice
Construction of pSB1C3-Promotor(J23111)-RBS-SRll-Htrll-Tar by Overlap extension PCR(we confirmed the sequence!!)

[Photocontrol] Preparation of plasmid
Constitutive promoter (BBa_23107)
Construct of [Constitutive promoter-RBS-ho1-RBS-PcyA-RBS-GlrN-Trem-OmpR promoter-RBS-GFP-Trem]

[Aggregation] Construction of constitutive promoter-RBS-Antigen 43-Double terminator

October 4 - October 10 [Uptake] ・Subcloning of iron peptide-cording gene
(We amplified the gene cording C-terminus of alpha-synuclein with iron-binding capacity by PCR from pET28a having alpha-synuclein gene and. For subcloning we used Primers that contained NcoI or XhoI site. The PCR product with NcoI and XhoI site was confirmed by electrophoresis and purified from agarose gel.)

・Insertion of iron-binding peptide gene into pET28a vector
・Plasmid extraction and transformation of BL21(DE3)
・Measurement of iron-binding function

The cells were disrupted by sonication. The samples were centrifuged at 15,000 G for 20 min and corrected the supernatant. We measured Fe (III) concentration in the supernatant by color reaction using 1, 10- phenanthroline which is chelating agent of iron ion. When 1, 10- phenanthroline chelate iron ion, the absorbance at 510 nm increases. Expression of iron-binding peptide was confirmed by SDS-PAGE. We also carried out same experiments against cells transformed by pET28a as a control.

As a result of comparison with control cells, iron ion concentration in the supernatant did not change. Expression of iron-binding peptide was also failed.

[Tank] ・Construction of PH-pduABJKNU-terminator and PM-pduABJKNU-terminator
(2 promoters; PH→J23102, PM→J23107)
・Construction of PH-pduP-GFPuv-terminator, PM-pduP-GFPuv-terminator, PL-pduP-GFPuv-terminator
(3 promoters; PH→J23102, PM→J23107, PL→J23109)
[Aggregation] Evaluation
We Observed that antigen43 works successfully .

October 11 - October 17 [Uptake] Addition of promoter, RBS and terminator to iron-binding peptide gene
Insertion of [Promoter]-[RBS]-[iron-binding peptide]-[terminator] gene into pSB1C3 vector

[Tank] Sequence analysis of PH-pduABJKNU-terminator, PM-pduABJKNU-terminator, PH-pduP-GFPuv-terminator and PL-pduP-GFPuv-terminator
Subcloning of PH-pduABJKNU-terminator into pSB1C3
Subcloning of PH-pduP-GFPuv-terminator and PL-pduP-GFPuv-terminator into pSB1C3
Construction of PH-pduABJKNU-terminator-PH-pduP-GFPuv-terminator and PH-pduABJKNU-terminator-PL-pduP-GFPuv-terminator

[Phototaxis] &[Aggregation] Construction of pSB1K3-OmpR positive promoter-RBS-Antigen 43-Double terminator.

October 18 - October 23 [Uptake] We carried out sequence analysis of the construct ([Promoter]-[RBS]-[iron-binding peptide]-[terminator]),but we could not confirm the sequence.

[Phototaxis] Evaluation
We Observed that phototaxis device properly works.

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