Ymiw1.html

Week 1 ~ Week 4 (7/1~7/28)

Week 1 (7/1~7/7)

Sunday
* Group meeting for topic decision-Venus project/endosynbiosis
* preparation and cultivation of vector pSB1C3 & Ptrc-PNSII-Strep
-25c.c LB medium +25ul Strep + Ptrc-PNSII-Strep
-25c.c LB medium +25ul Cm + pSB1C3

Monday
SRB

      * Find for expertise and discuss with them to make a novel project:
NTU/Fujen U/Fu In U/Chung Shin U

• SQR

      * extraction of plasmid DNA(pSB1C3 & Ptrc-PNSII-Strep)
* extract plasmid DNA of Synechococcus elongatus PCC 7002
(a kind of cyanobacteria)
* waiting for primer of SQR


Tuesday
SRB

      * bacteria choosing: purple sulfur bacteria/ hot spring bacteria / sulfur reducing bacteria
* Figure out the biochemistry characteristic of Desufulvibrio Defulficans

• SQR(Cloning of SQR from PCC7002 begin)

      * PCR of SQR
Template: plasmid DNA of sp.PCC7002
Forward primer: SQR-FP (EX_SQR_F)
Reverse primer: SQR-RP (S_SQR_R)
conclusion: positive
*PCR clean up
*enzyme cut
Rescrition enzyme: pSB1C3: EcoRI.SpeI
Ptrc-PNSII-Strep: EcoRI.XbaI
SQR: EcoRI.SpeI


Wednesday
SRB

      * Figure out the biochemistry characteristic of Desufulvibrio Defulficans

• SQR

      * Clean up
* Ligation: pSB1C3-SQR & Ptrc-PNSII-Strep-SQR
conclusion: positive
* Transformation of competent cell
-prepare Cm20 & Strep plate
- Plate the bacteria onto plates and place at 37oC overnight


Thursday
SQR

      * acquire many colony on the plate
-pick 8 colony on each plate(Cm1~Cm8&Strep1~Strep8)
* Colony PCR
Forward primer: VF2
Reverse primer: SQR-RP
Conclusion: Cm1~Cm8 no band
Strep1~Strep8 : positive
Cm1~Cm8 colony PCR again
Conclusion: positive

Friday
SQR

      * Enzyme check
-pSB1C3-SQR
Enzyme: NotI-HF (1307+2406)
Conclusion: positive
-Ptrc-PNSII-Strep-SQR
Enzyme: KpnI (3137+2679)
Conclusion: positive


Saturday
SQR

      * Send for sequencing
Conclusion: positive

Week 2 (7/8~7/14)



Sunday
SRB

      * Set up whole artificial "sulfur" metabolism pathway

• SQR

      * Prepare for chemical compound (order DCMU, Na2S)
Monday

• SRB

      * Set up whole artificial "sulfur" metabolism pathway

• SQR

* Transform Ptrc-PNSII-Strep-SQR into cyanbacteria (sp. PCC 7942)
* The transformation will take 2 weeks


Tuesday
SRB

      * Order for bacteria from BCRC: SRB- Desufulvibrio Desuficans ATCC #27774

• SmtA (Cloning of SmtA from PCC7942 begin)

      * PCR of SmtA
Template: plasmid DNA of sp.PCC7942
Forward primer: SmtA-FP (EX_ SmtA _F)
Reverse primer: SmtA -RP (S_ SmtA _R)
conclusion: positive
*PCR clean up
*enzyme cut
Rescrition enzyme: pSB1C3: EcoRI.SpeI
SmtA: EcoRI.SpeI


Wednesday
SRB

     * Meeting with Prof. chu ( researcher in sinica academic,the top research institute in Taiwan) topic: 1.photosynthesis pathway I and II 2. Replace the H2O function with H2S inside the whole photosynthesis pathway

• smtA

      * Clean up
* Ligation: pSB1C3-SmtA
conclusion: positive
* Transformation of competent cell
-prepare Cm20 plate
- Plate the bacteria onto plates and place at 37oC overnight

Thursday
SRB

     * Meeting with Prof. chu ( researcher in sinica academic,the top research institute in Taiwan) topic: 1.photosynthesis pathway I and II 2. Replace the H2O function with H2S inside the whole photosynthesis pathway

• SmtA

      * acquire many colony on the plate
-pick 8 colony on each plate(Cm1~Cm8)
* Colony PCR
Forward primer: CmR gene-R-SpeI
Reverse primer: SmtA-RP
Conclusion: Cm1.Cm2.Cm4.Cm5.Cm6.Cm8 positive

Friday
SRB

      * SRB genome blast : Desufulvibrio Defulficans/ Desulculvibrio gigas/ Desufulvibrio Vulgas

• SmtA

      * Enzyme check
-pSB1C3- SmtA
Enzyme: AgeI-HF + NcoI-HF (1350+893)
Conclusion: positive

Saturday
* Send for sequencing
Conclusion: positive

 

Week 3(7/15~7/21)

Sunday
SRB

      * Prepare special anaerobic medium for Desufulvibrio ssp. ( several pro it'll ingredient kindly from NTU Prof. Lin, expertise in SRB)

Monday
SRB

      * Culture for anaerobe bacteria- sulfur reducing bacteria: Desufulvibrio Desulficans


Tuesday
SRB

      * Culture for anaerobe bacteria- sulfur reducing bacteria: Desufulvibrio Desulficans

• Invasin+LLO

      * Ask for invasion+llo on Ptrc-PNSIII-Amp from Harvard prof. Pamela A. Silver

Wednesday
SRB

      * Gene design for cloning: whole genome data from NCBI, specific enzyme chosen from KEGG- Adenylyl sulfate reductase, sulfate Adenylyl transferase, pyrophosphatase, sulfur reductase

Thursday
SRB

      * cloning site decision for cloning: 5':MfeI,XbaI. 3':SbfI, SpeI. Middle: BamHI


Friday
* A big typhoon is coming!! (exp delayed for two days!!)


Saturday
* A big typhoon is coming!! (exp delayed for two days!!)

Week 4(7/22~7/28)


Sunday
SRB

      * Subculture for Desufulvibrio Desuficans (fail 1st time)


Monday
SRB

      * Subculture for Desufulvibrio Desuficans (fail 2nd time)

• Invasin+LLO

      * The invasion +llo on Ptrc-PNSIII-AMP from professor in Harvard finally come!!!
* Enzyme check
- Ptrc-PNSIII-AMP-invasion +llo
Enzyme: EcoRI-HF (2006+7437)
Conclusion: positive


Tuesday
SRB

      * Looking for technique support for anaerobe bacteria culture: NTU and BCRC

• Invasin+LLO

      * Send for sequencing
Conclusion: positive


Wednesday
SRB

      * Go sinica  academic  ( the top research constitute in Taiwan ) and make a discussion about synthetic biology with prof. Wu ( interested in ecology, Cyanobacteria and species diversity research)


Thursday
SRB

      * Go sinica academic and make a discussion about synthetic biology with prof. Wu ( interested in ecology, Cyanobacteria and species diversity research)


Saturday
Human practice

      * Human practice: thinking twice about that whether human beings have right to create a new organism? ( to become God and change the world)