Team:NYMU-Taipei/ymiw2.html

Week 5 ~ Week 8 (7/29~8/25)

Week 5 (7/29~8/4)

Sunday
SRB

      * Other alternatives decision: find out specific enzyme for sulfur reducing from KEGG, paper survey


Monday
SRB

      * Paper survey (waiting for primer also)
* Design primer for NosZ and NorCB


Tuesday
SRB(Cloning ofCycI)

      * PCR construction for new pSB1C3 (we named it MF-pSB1C3): MfeI-Xba I-pSB1C3 backbone- Sbf I-Spe I ( for tater gene which has endogenous EcoRI, PstI cutting site)
* PCR of CyC I
Template DNA
Forward primer: CyC I -FP (MX_ SmtA _F)
Reverse primer: CyC I -RP (SbfIS_ SmtA _R)
conclusion: positive
*PCR clean up
*enzyme cut
Rescrition enzyme: pSB1C3: MfeI.SbfI
SmtA: MfeI.SbfI

• NosZ+NorCB

      * PCR of NosZ
Template DNA: pseudomonas aeruginosa
Forward primer: NosZ -FP (EX_ NosZ _F)
Reverse primer: NosZ -RP (SbfIS_ NosZ _R)
conclusion: positive
*PCR clean up
*enzyme cut
Rescrition enzyme: MF-pSB1C3: EcoRI. SbfI
NosZ: EcoRI. SbfI
* PCR of NorCB
Template DNA
Forward primer: NorCB -FP (EX _ NorCB _F)
Reverse primer: NorCB -RP (SbfIS_ NorCB _R)
conclusion: positive
*PCR clean up
*enzyme cut
Rescrition enzyme: MF-pSB1C3: EcoRI. SbfI
NorCB: EcoRI. SbfI
To combine NosZ and NorCB, we need to do another enzyme cut
*enzyme cut
Rescrition enzyme: MF-pSB1C3: EcoRI. SbfI
NosZ: EcoRI. SpeI
NorCB: XbaI. SbfI


Wednesday
SRB

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

• NosZ+NorCB

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

Thursday
 SRB

      * acquire many colony on the plate
-pick 8 colony on each plate(Cm1~Cm8)
* Colony PCR
Forward primer: CmR gene-R-SpeI
Reverse primer: CycI-RP
Conclusion: Cm1~ Cm 8 positive

• NosZ+NorCB

      * acquire some colony( <=8 ) on the plate
- pick 8 colony on MF-pSB1C3-NosZ plate(Cm1~Cm8)
- pick 6 colony on MF-pSB1C3-NorCB plate(Cm1~Cm6)
- pick 8 colony on MF-pSB1C3-NosZ-NorCB plate(Cm1~Cm8)
* Colony PCR
Forward primer: CmR gene-R-SpeI
Reverse primer(for MF-pSB1C3-NosZ): NosZ –RP
Conclusion: Cm1~ Cm 8 positive
Reverse primer(for MF-pSB1C3-NorCB): NorCB –RP
Conclusion: Cm2 & Cm 4 positive
Reverse prime(for MF-pSB1C3-NosZ-NorCB)r: NorCB -RP
Conclusion: Cm1. Cm 5. Cm6 positive

Friday
SRB

      * Enzyme check
-MF-pSB1C3- CycI
Enzyme: XbaI + SpeI (2157+2938)
Conclusion: positive

• NosZ+NorCB

      * Enzyme check
MF-pSB1C3-NosZ
Enzyme: XbaI + SpeI (2157+2938)
Conclusion: positive
MF-pSB1C3-NorCB
Enzyme: XbaI + SpeI (2157+2938)
Conclusion: positive
MF-pSB1C3-NosZ-NorCB
Enzyme: XbaI + SpeI (2157+2938)
Conclusion: positive


Saturday
SRB

      * Send for sequencing
Conclusion: positive

•    NosZ+NorCB

      * Send for sequencing
Conclusion: positive



Week 6 (8/5~8/11)



Sunday
SRB

      * Failure to activate dried Desufulvibrio Desulficans ( ask for help from BCRC)

• NirN+NirS

      * PCR of NirN
Template DNA: pseudomonas aeruginosa
Forward primer: NirN -FP (EX_ NirN _F)
Reverse primer: NirN -RP (BamHI_ NirN _R)
conclusion: positive
*PCR clean up

* PCR of NirS
Template DNA: pseudomonas aeruginosa
Forward primer: NirS -FP (BamHI_ NirS _F)
Reverse primer: NirS -RP (PS_ NirS _R)
conclusion: positive
*PCR clean up
*enzyme cut
Rescrition enzyme: pSB1C3: EcoRI.SpeI
NirN: EcoRI.BamHI
NirS: BamHI.SpeI


Monday
SRB

      * sub clone CyC I gene from MfeI- XbaI-pSB1C3-SbfI-SpeI-CyC I into pTrc-kan plasmid( GE lab constructed plasmid for Cyanobacteria transgene technique, site #1)
* Ptrc-kan cut with EcoRI, XbaI, #4 bfr, while MfeI-XbaI-pSB1C3-SbfI-SpeI- CyC I cut with, MfeI, Sbf I, #4 (smart trick : MfeI and EcoRI form scar, SbfI and XbaI form scar, in order to prevent endogenous EcoRI and PstI site)

• NirN+NirS

      * Ligatiom, transform with kan solid agar plate
* Clean up
* Ligation: pSB1C3-NirN-RBS-NirS
conclusion: positive
* Transformation of competent cell
-prepare Cm20 plate
- Plate the bacteria onto plates and place at 37oC overnight

Tuesday
SRB

      * Colony PCR to check whether pTrc-kan-CyC I is correct and amplification the bacteria colony
* acquire many colony on the plate
-pick 8 colony on each plate(Kan1~Kan8)
* Forward primer: VF2
Reverse primer: CycI-RP
Conclusion: Kan1~ Kan 4 positive

• NirN+NirS

      * acquire many colony on the plate
-pick 8 colony on each plate(Cm1~Cm8)
* Colony PCR
Forward primer: CmR gene-R-SpeI
Reverse primer: NirS-RP
Conclusion: Cm2~ Cm 6.Cm8 positive


Wednesday
SRB

      * Enzyme check
-MF-pSB1C3- CycI
Enzyme: MfeI + SpeI (1307+2406)
Conclusion: positive

• NirN+NirS

      * Enzyme check
pSB1C3- NirN-NirS
Enzyme: MfeI + SpeI (1307+2406)
Conclusion: positive


Thursday
SRB

      * Paper survey for hot spring bacteria and purple sulfur bacteria, to double check whether they are better choices for our project to go on? Or we should still use Sulfur reducing bacteria

• NirN+NirS

      * Send for sequencing
Conclusion: positive

Friday
SRB

      * BCRC finish the bacteria type analysis about Desufulvibrio Desulficans and ready to amplifies this SRB for us (thanks to God!!)


Saturday
SRB

      * Paper survey for hot spring bacteria and purple sulfur bacteria, to double check whether they are better choices for our project to go on? Or we should still use Sulfur reducing bacteria
* Start to prepare for the Taiwan iGEMers’ fest (We are the host)

Week 7 (8/12~8/18)

Sunday
      * Prepare for the Taiwan iGEMers’ fest(exp delayed for three days!!)


Monday
      * Prepare for the Taiwan iGEMers’ fest(exp delayed for three days!!)


Tuesday
      * Prepare for the Taiwan iGEMers’ fest(exp delayed for three days!!)


Wednesday
      * Taiwan iGEMers’ fest(NYMU-Taipei.NTU-Taida. NCTU_Formosa)


Thursday
      * Discuss the advantages and disadvantages of our project after Taiwan iGEMers’ fest.
      * Get some rest


Friday
      * Paper survey for hot spring bacteria and purple sulfur bacteria, to double check whether they are better choices for our project to go on? Or we should still use Sulfur reducing bacteria


Saturday
      * Primer design for sulfite reductase in Desufulvibrio Desulficans: DsrI, DsrII
      * Waiting for primers to come

Week 8 (8/19~8/25)

Sunday
SRB

      * PCR of DsrI and DsrII
Template: genomic DNA from  Desufulvibrio Desulficans
For DsrI:
Forward primer: DsrI -FP (MX_ DsrI _F)
Reverse primer: DsrI -RP (BamHI _ DsrI _R)
conclusion: positive
For DsrII:
Forward primer: DsrII -FP (BamHI_DsrI _F)
Reverse primer: DsrII -RP (SbfI-SpeI_DsrII _R)
*PCR clean up
*enzyme cut
Rescrition enzyme: MF-pSB1C3: MfeI.SpeI
DsRI-BamHI-DsrII: MfeI.SpeI

• NosZ-NorCB

      * sub clone NosZ+NorCB gene from pSB1C3-NosZ-NorCB into pTrc-Amp plasmid( GE lab constructed plasmid for Cyanobacteria transgene technique, site #3)
* Ptrc-Amp cut with EcoRI, XbaI, #4 bfr, while pSB1C3-NosZ-NorCB cut with, EcoRI, Spe I

 

Monday
SRB

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

• NosZ-NorCB

      * Ligatiom, transform with kan solid agar plate
* Clean up
* Ligation: pSB1C3-NosZ-NorCB
conclusion: positive
* Transformation of competent cell
-prepare Amp20 plate
- Plate the bacteria onto plates and place at 37oC overnight

• SQR

      * Synechococcus sp. PCC7002 was cultured in the medium A2, a modification of Stevens et al. (1973) on a rotary shaker (100 rpm). The initial concentration of cells is controlled to an OD730 of 0.1 in fresh medium. Different concentration of sodium sulfide is added, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was put into experimental group. Cells are grown in the six-well-plates and monitored under OD730 every 24 hour.


Tuesday
SRB

      * acquire many colony on the plate
-pick 8 colony on each plate(Cm1~Cm8)
* Colony PCR
Forward primer: VF2
Reverse primer: DsrII-RP
Conclusion: Cm 3 &Cm 5 positive

• NosZ-NorCB

      * acquire many colony on the plate
-pick 8 colony on each plate(Amp 1~ Amp 8)
* Colony PCR
Forward primer: VF2
Reverse primer: NorCB-RP
Conclusion: Amp 1~ Amp 5 positive

• SQR

      * SQR part experiment


Wednesday
SRB

      * Enzyme check
MF-pSB1C3- DsrI-BamHI-DsrII
Enzyme: XbaI + SpeI (1307+2406)
Conclusion: positive

• NosZ-NorCB

* Enzyme check
pSB1C3-NosZ-NorCB
Enzyme: XbaI + SpeI (1307+2406)
Conclusion: positive

• SQR

      * SQR part experiment


Thursday
SRB

      * Send for sequencing
Conclusion: positive

• NosZ-NorCB

* Send for sequencing
Conclusion: positive

• SQR

      * SQR part experiment


Friday
SRB

      * Transfer Ptrc-PNSI-Kan- CyC I gene into cyanbacteria (sp. PCC 7942)

• NosZ-NorCB

      * Transfer Ptrc -PNSIII-Amp- NosZ-NorCB gene into cyanbacteria (sp. PCC 7942)

• SQR

      * SQR part experiment


Saturday
SRB

      * Microscopy analysis of Desufulvibrio Desulficans

• SQR

      * SQR part experiment