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

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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 July 26 ~August 1
 [solubilization]
-Culturing of pseudomonas aeruginosa
+Culturing of pseudomonas aeruginosa<br>
-TA cloning of RhlA and RhlB genes from pseudomonas aeruginosa
+TA cloning of RhlA and RhlB genes from pseudomonas aeruginosa<br><br>
 [Tank]
-Purchased Citrobacter freundii (Braak1928) Werkman and Gillen 1932
+Purchased Citrobacter freundii (Braak1928) Werkman and Gillen 1932<br><br>
 August 2 - August 8
 [solubilization]
-We carried out sequences analysis of RhlA and RhlB, but we could not confirm the sequence.
+We carried out sequences analysis of RhlA and RhlB, but we could not confirm the sequence.<br><br>
 [Tank]
-Amplification of the genes (pduAB, pduJK, pduN, pduU) respectively by PCR from Citrobacter freundii (Braak1928)
+Amplification of the genes (pduAB, pduJK, pduN, pduU) respectively by PCR from Citrobacter freundii (Braak1928)<br><br>
 August 9 - August 15
 [Tank]
-The PCR product (pduAB, pduJK, pduN, pduU) were inserted into pSB vector.
+The PCR product (pduAB, pduJK, pduN, pduU) were inserted into pSB vector.<br>
-DH5 were transformed by the constructed plasmids.
+DH5 were transformed by the constructed plasmids.<br>
-Colony PCR was carried out to confirm the constructions.
+Colony PCR was carried out to confirm the constructions.<br>
-Extraction of plasmids.
+Extraction of plasmids.<br>
-Sequence analysis of cloned genes
+Sequence analysis of cloned genes<br>
-・Analyzed the sequence of last-week extracted plasmids
+<ul>
+<li>・Analyzed the sequence of last-week extracted plasmids</li>
 ・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.
+</ul><br>
 August 16 - August 22
 [solubilization]
-TA cloning of RhlA and RhlB genes from pseudomonas aeruginosa again
+TA cloning of RhlA and RhlB genes from pseudomonas aeruginosa again<br>
-Subcloning of RhlA and RhlB
+Subcloning of RhlA and RhlB<br>
-Insertion of RhlA and RhlB genes into pSB vector
+Insertion of RhlA and RhlB genes into pSB vector<br><br>
 [Tank]
-pduAB and pduJK  genes have Pst1 sites. So we removed the Pst1 sites in pduAB and pduJK by overlap PCR
+pduAB and pduJK  genes have Pst1 sites. So we removed the Pst1 sites in pduAB and pduJK by overlap PCR<br><br>
 August 23 - August 29
 [Tank]
-Confirmation of modification of Pst1 site in pduAB and pduJK by sequence analysis
+Confirmation of modification of Pst1 site in pduAB and pduJK by sequence analysis<br>
-Amplification of pduAB, pduJK, pduN, pduU genes using primers which attached RBS(B0034) to each gene
+Amplification of pduAB, pduJK, pduN, pduU genes using primers which attached RBS(B0034) to each gene<br><br>
 August 30 - September 5
 [solubilization]
-Construction of promoter-RBS-RhlA-term and promoter-RBS-RhlB-term parts using megaprimer made by overlap PCR.
+Construction of promoter-RBS-RhlA-term and promoter-RBS-RhlB-term parts using megaprimer made by overlap PCR.<br>
-The PCR product (promoter-RBS-RhlA-term and promoter-RBS-RhlB-term) was inserted into pSB vector
+The PCR product (promoter-RBS-RhlA-term and promoter-RBS-RhlB-term) was inserted into pSB vector<br>
-Transformation of E. coli DH5 by the plasmid (We did not get white colony)
+Transformation of E. coli DH5α by the plasmid (We did not get white colony)<br><br>
 [Tank]
-Construction of pduABJK by 3A assembly
+Construction of pduABJK by 3A assembly <br>
-Construction of pduU-terminator
+Construction of pduU-terminator<br><br>
 [Phototaxis]
-Overlap extension PCR & Sequence analysis
+Overlap extension PCR & Sequence analysis<br>
-failed→SRll-Htrll-Tar
+failed→SRll-Htrll-Tar<br>
-succeed→Htrll (pSB1C3-Htrll sequence was comfirmed)
+succeed→Htrll (pSB1C3-Htrll sequence was comfirmed)<br><br>
 [Photocontrol]
-Preparation of plasmid
+Preparation of plasmid<br>
-    OmpR promoter (BBa_R0082)
+    OmpR promoter (BBa_R0082)<br>
-    RBS-GFP-Term
+    RBS-GFP-Term<br>
-Construction of OmpR-promoter-RBS-GFP-Term by 3A assembly
+Construction of OmpR-promoter-RBS-GFP-Term by 3A assembly<br>
-Sequence analysis of OmpR-promoter-RBS-GFP-Term (we could not confirm the sequence.)
+Sequence analysis of OmpR-promoter-RBS-GFP-Term (we could not confirm the sequence.)<br><br>
 [Aggregation]
-TA-cloning of Antigen 43
+TA-cloning of Antigen 43<br>
-pGEMT-Antigen 43(Wildtype) was constructed
+pGEMT-Antigen 43(Wildtype) was constructed<br><br>
 September 6 - September 12
 [Tank]
-Construction of pduNU-terminator
+Construction of pduNU-terminator<br><br>
 [Phototaxis]
-Construct of pSB1C3-SRll-Htrll-Tarm
+Construct of pSB1C3-SRll-Htrll-Tarm<br><br>
 [Photocontrol]
-Construction of OmpR-promoter-RBS-GFP-Term again.
+Construction of OmpR-promoter-RBS-GFP-Term again.<br>
-Sequence analysis of OmpR-promoter-RBS-GFP-Term (we confirmed the sequence!!)
+Sequence analysis of OmpR-promoter-RBS-GFP-Term (we confirmed the sequence!!)<br><br>
 [Aggregation]
 Subcloning of Antigen 43(Wildtype)
-  pSB1C3-Antigen 43(Wild type) was constructed.
+  pSB1C3-Antigen 43(Wild type) was constructed.<br>
-Removal of the 6 PstI sites in Antigen 43(Wild type) by PCR.
+Removal of the 6 PstI sites in Antigen 43(Wild type) by PCR.<br><br>
 September 13 - September 19
 [Tank]
-Construction of pduABJKNU-terminator
+Construction of pduABJKNU-terminator<br><br>
 [Phototaxis]
-Sequence analysis
+Sequence analysis<br>
-	pSB1C3-Tar, pSB1C3-SRll and pSB1C3-SRll-Htrll-Tar sequence were comfirmed
+	pSB1C3-Tar, pSB1C3-SRll and pSB1C3-SRll-Htrll-Tar sequence were comfirmed<br><br>
 [Photocontrol]
-Preparation of the plasmid
+Preparation of the plasmid<br>
-     RBS-ho1-RBS-PcyA-RBS-GlrN-Trem
+     RBS-ho1-RBS-PcyA-RBS-GlrN-Trem<br>
-Construct of RBS-ho1-RBS-PcyA-RBS-GlrN-Trem- OmpR promoter-RBS-GFP-Trem
+Construct of RBS-ho1-RBS-PcyA-RBS-GlrN-Trem- OmpR promoter-RBS-GFP-Trem<br><br>
 [Aggregation]
-PstI sites modified-Antigen 43 gene was inserted into pSB1C3.
+PstI sites modified-Antigen 43 gene was inserted into pSB1C3.<br>
-Construct of pSB1K3-RBS-Antigen 43
+Construct of pSB1K3-RBS-Antigen 43<br><br>
 September 20 - September 26
-[Tank]
-待ち
 [Phototaxis]
-Construction of pSB1C3-RBS-SRll-Htrll-Tar by 3A assembly (sequence couldn’t be comfirmed)
+Construction of pSB1C3-RBS-SRll-Htrll-Tar by 3A assembly (sequence couldn’t be comfirmed)<br><br>
 [Photocontrol]
-Construct of RBS-ho1-RBS-PcyA-RBS-GlrN-Trem- OmpR promoter-RBS-GFP-Trem again
+Construct of RBS-ho1-RBS-PcyA-RBS-GlrN-Trem- OmpR promoter-RBS-GFP-Trem again<br>
-Sequence analysis of the construct (we confirmed the sequence!!)
+Sequence analysis of the construct (we confirmed the sequence!!)<br><br>
 [Aggregation]
-Construct of pSB1K3-RBS-Antigen 43 Double terminator
+Construct of pSB1K3-RBS-Antigen 43 Double terminator<br><br>
 September 27 - October 3
 [Tank]
-Construction of pduP-GFPuv-terminator
+Construction of pduP-GFPuv-terminator<br><br>
 [Phototaxis]
-Construction of pSB1C3-RBS-SRll-Htrll-Tar by 3A assembly (sequence couldn’t be comfirmed) twice
+Construction of pSB1C3-RBS-SRll-Htrll-Tar by 3A assembly (sequence couldn’t be comfirmed) twice<br>
-Construction of pSB1C3-Promotor(J23111)-RBS-SRll-Htrll-Tar by Overlap extension PCR(we confirmed the sequence!!)
+Construction of pSB1C3-Promotor(J23111)-RBS-SRll-Htrll-Tar by Overlap extension PCR(we confirmed the sequence!!)<br><br>
 [Photocontrol]
-Preparation of plasmid
+Preparation of plasmid<br>
-     Constitutive promoter (BBa_23107)
+     Constitutive promoter (BBa_23107)<br>
-Construct of [Constitutive promoter-RBS-ho1-RBS-PcyA-RBS-GlrN-Trem-OmpR promoter-RBS-GFP-Trem]
+Construct of [Constitutive promoter-RBS-ho1-RBS-PcyA-RBS-GlrN-Trem-OmpR promoter-RBS-GFP-Trem]<br><br>
 [Aggregation]
-Construction of constitutive promoter-RBS-Antigen 43-Double terminator
+Construction of constitutive promoter-RBS-Antigen 43-Double terminator <br><br>
 October 4 - October 10
 [Uptake]
-・Subcloning of iron peptide-cording gene
+・Subcloning of iron peptide-cording gene<br>
-(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.)
+(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.)<br><br>
-・Insertion of iron-binding peptide gene into pET28a vector
+・Insertion of iron-binding peptide gene into pET28a vector<br>
-・Plasmid extraction and transformation of BL21(DE3)
+・Plasmid extraction and transformation of BL21(DE3)<br>
-・Measurement of iron-binding function
+・Measurement of iron-binding function<br><br>
-<Method> 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.
-<Results> 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.
+<Method> 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.<br><br>
+<Results> 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.<br><br>
 [Tank]
-・Construction of PH-pduABJKNU-terminator and PM-pduABJKNU-terminator
+・Construction of PH-pduABJKNU-terminator and PM-pduABJKNU-terminator<br>
-(2 promoters; PH→J23102, PM→J23107)
+(2 promoters; PH→J23102, PM→J23107)<br>
-・Construction of PH-pduP-GFPuv-terminator, PM-pduP-GFPuv-terminator, PL-pduP-GFPuv-terminator
+・Construction of PH-pduP-GFPuv-terminator, PM-pduP-GFPuv-terminator, PL-pduP-GFPuv-terminator<br>
-(3 promoters; PH→J23102, PM→J23107, PL→J23109)
+(3 promoters; PH→J23102, PM→J23107, PL→J23109)<br>
 [Aggregation]
-Evaluation
+Evaluation<br>
-We Observed that antigen43 works successfully .
+We Observed that antigen43 works successfully .<br><br>
 October 11 - October 17
 [Uptake]
-Addition of promoter, RBS and terminator to iron-binding peptide gene
+Addition of promoter, RBS and terminator to iron-binding peptide gene<br>
-Insertion of [Promoter]-[RBS]-[iron-binding peptide]-[terminator] gene into pSB1C3 vector
+Insertion of [Promoter]-[RBS]-[iron-binding peptide]-[terminator] gene into pSB1C3 vector<br><br>
 [Tank]
-Sequence analysis of PH-pduABJKNU-terminator, PM-pduABJKNU-terminator, PH-pduP-GFPuv-terminator and PL-pduP-GFPuv-terminator
+Sequence analysis of PH-pduABJKNU-terminator, PM-pduABJKNU-terminator, PH-pduP-GFPuv-terminator and PL-pduP-GFPuv-terminator<br>
-Subcloning of PH-pduABJKNU-terminator into pSB1C3
+Subcloning of PH-pduABJKNU-terminator into pSB1C3<br>
-Subcloning of PH-pduP-GFPuv-terminator and PL-pduP-GFPuv-terminator into pSB1C3
+Subcloning of PH-pduP-GFPuv-terminator and PL-pduP-GFPuv-terminator into pSB1C3<br>
-Construction of PH-pduABJKNU-terminator-PH-pduP-GFPuv-terminator and PH-pduABJKNU-terminator-PL-pduP-GFPuv-terminator
+Construction of PH-pduABJKNU-terminator-PH-pduP-GFPuv-terminator and PH-pduABJKNU-terminator-PL-pduP-GFPuv-terminator<br><br>
 [Phototaxis] &[Aggregation]
-Construction of pSB1K3-OmpR positive promoter-RBS-Antigen 43-Double terminator.
+Construction of pSB1K3-OmpR positive promoter-RBS-Antigen 43-Double terminator.<br><br>
 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.
+We carried out sequence analysis of the construct ([Promoter]-[RBS]-[iron-binding peptide]-[terminator]),but we could not confirm the sequence.<br><br>
 [Phototaxis]
-Evaluation
+Evaluation<br>
-We Observed that phototaxis device properly works.
+We Observed that phototaxis device properly works.<br><br>
+</body>
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