Team:Tokyo-NoKoGen/experiments/Lab note
From 2010.igem.org
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pSB1C3-Antigen 43(Wild type) was constructed.<br> | pSB1C3-Antigen 43(Wild type) was constructed.<br> | ||
Removal of the 6 PstI sites in Antigen 43(Wild type) by PCR.<br><br> | Removal of the 6 PstI sites in Antigen 43(Wild type) by PCR.<br><br> | ||
+ | |||
+ | <h3>[Lysis]</h3> | ||
+ | Construction of BBa_K317037 and BBa_K317038<br> | ||
+ | |||
<h2>September 13 - September 19</h2> | <h2>September 13 - September 19</h2> | ||
Line 111: | Line 115: | ||
PstI sites modified-Antigen 43 gene was inserted into pSB1C3.<br> | PstI sites modified-Antigen 43 gene was inserted into pSB1C3.<br> | ||
Construct of pSB1K3-RBS-Antigen 43<br><br> | Construct of pSB1K3-RBS-Antigen 43<br><br> | ||
+ | |||
+ | |||
+ | <h3>[Lysis]</h3> | ||
+ | Construction of BBa_K317037 and BBa_K317038<br> | ||
+ | |||
<h2>September 20 - September 26</h2> | <h2>September 20 - September 26</h2> | ||
Line 138: | Line 147: | ||
<h3>[Aggregation]</h3> | <h3>[Aggregation]</h3> | ||
Construction of constitutive promoter-RBS-Antigen 43-Double terminator <br><br> | Construction of constitutive promoter-RBS-Antigen 43-Double terminator <br><br> | ||
+ | |||
+ | <h3>[Lysis]</h3> | ||
+ | Construction of BBa_K317039 and BBa_K317042<br> | ||
+ | |||
<h2>October 4 - October 10</h2> | <h2>October 4 - October 10</h2> | ||
Line 161: | Line 174: | ||
Evaluation<br> | Evaluation<br> | ||
We Observed that antigen43 works successfully .<br><br> | We Observed that antigen43 works successfully .<br><br> | ||
+ | |||
+ | |||
+ | <h3>[Lysis]</h3> | ||
+ | Construction of BBa_K317040<br> | ||
<h2>October 11 - October 17</h2> | <h2>October 11 - October 17</h2> | ||
Line 182: | Line 199: | ||
<h3>[Phototaxis]</h3> | <h3>[Phototaxis]</h3> | ||
Evaluation<br> | Evaluation<br> | ||
- | We | + | We observed that phototaxis device properly works.<br><br> |
+ | |||
+ | <h3>[Lysis]</h3> | ||
+ | Evaluation of BBa_K317039 and BBa_K317040<br> | ||
+ | We observed that lysis device properly works.<br><br> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 03:44, 28 October 2010
July 26 ~August 1
[solubilization]
Culturing of pseudomonas aeruginosaTA cloning of RhlA and RhlB genes from pseudomonas aeruginosa
[Tank]
Purchased Citrobacter freundii (Braak1928) Werkman and Gillen 1932August 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.DH5α 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 againSubcloning 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 PCRAugust 23 - August 29
[Tank]
Confirmation of modification of Pst1 site in pduAB and pduJK by sequence analysisAmplification 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 assemblyConstruction of pduU-terminator
[Phototaxis]
Overlap extension PCR & Sequence analysisfailed→SRll-Htrll-Tar
succeed→Htrll (pSB1C3-Htrll sequence was comfirmed)
[Photocontrol]
Preparation of plasmidOmpR 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 43pGEMT-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.
[Lysis]
Construction of BBa_K317037 and BBa_K317038September 13 - September 19
[Tank]
Construction of pduABJKNU-terminator[Phototaxis]
Sequence analysispSB1C3-Tar, pSB1C3-SRll and pSB1C3-SRll-Htrll-Tar sequence were comfirmed
[Photocontrol]
Preparation of the plasmidRBS-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
[Lysis]
Construction of BBa_K317037 and BBa_K317038September 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 againSequence analysis of the construct (we confirmed the sequence!!)
[Aggregation]
Construct of pSB1K3-RBS-Antigen 43 Double terminatorSeptember 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) twiceConstruction of pSB1C3-Promotor(J23111)-RBS-SRll-Htrll-Tar by Overlap extension PCR(we confirmed the sequence!!)
[Photocontrol]
Preparation of plasmidConstitutive 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[Lysis]
Construction of BBa_K317039 and BBa_K317042October 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
[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]
EvaluationWe Observed that antigen43 works successfully .
[Lysis]
Construction of BBa_K317040October 11 - October 17
[Uptake]
Addition of promoter, RBS and terminator to iron-binding peptide geneInsertion 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-terminatorSubcloning 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]
EvaluationWe observed that phototaxis device properly works.
[Lysis]
Evaluation of BBa_K317039 and BBa_K317040We observed that lysis device properly works.