Ymiw3.html

Week 9 ~ Week 11 (8/26~9/15)

Week 9 (7/29~8/4)

Sunday
SRB

      * Sub clone DsrI-BamHI-DsrII gene from pSB1C3 into Ptrc-kan plasmid
* Ptrc-kan plasmid cut with EcoRI, XbaI
* MF-pSB1C3 -DsrI-BamHI-DsrII gene Cut with MfeI, SpeI
*(smart trick: MfeI and EcoRI form scar, SbfI and SpeI form scar, to prevent endogenous EcoRI and PstI site)

• SQR

* Synechococcus sp. PCC7002 was cultured in the medium A2, rotary shaker and initial cell concentration are the same as the last experiment. DCMU is diluted with A2 medium into different concentration, and 10 mM of sodium sulfide is added to the experimental group. The measurement method and frequency of cell growth has been described previously.


Monday
SRB

      * Ligation, transformation pTrc-kan-DsrI-BamHI-DsrII plasmid into DH5alpha competent cell, cultured with Kan solid agar plate

• SQR

      * SQR part experiment


Tuesday
SRB

      * colony PCR and enzyme check to pick up correct colonies
* acquire many colony on the plate
-pick 8 colony on each plate(Kan 1~ Kan 8)
* Forward primer: VF2
Reverse primer: DsrII-RP
Conclusion: Kan 1 & Kan 6~8 positive

• SQR

      * SQR part experiment


Wednesday
SRB

      * Send for sequencing
Conclusion: positive

• SQR

      * SQR part experiment


Thursday
SRB

      * Function assay with pTrc-kan-CyC I gene transformed E.Coli
* Transfer pTrc-kan-DsrI-BamHI-DsrII gene into genomic DNA of Cyanobacteria

• SQR

      * SQR part experiment


Friday
SRB

      * Cyanobacteria CyC I gene stable clone established (wish we can establish cyanobacteria DsrI-BamHI-DsrII stable clone too!!)

• SQR

      * SQR part experiment


Saturday
SQR

      * SQR part experiment



Week 10 (9/2~9/8)


Sunday
NapABC

      * primer design for NapABC and new backbone MS-pSB1C3

• SQR

      * With the result of previous experiment, the concentration of both sodium sulfide and DCMU is adjusted. Sodium sulfide is diluted from 40mM to 2.5mM, while DCMU 0.5 μM is added into the experimental group.


Monday
* PCR construction for new pSB1C3 (we named it MS-pSB1C3, different from pSB1C3 and MF-pSB1C3): MfeI-Xba I-pSB1C3 backbone- Pst I-Spe I
* PCR of NapABC
Template DNA: pseudomonas aeruginosa
Forward primer: NapABC -FP (MX_ NapABC _F)
Reverse primer: NapABC -RP (PS_ NapABC _R)
conclusion: positive
*PCR clean up
*enzyme cut
Rescrition enzyme: MS-pSB1C3: MfeI.SpeI
NapABC: MfeI.SpeI

• SQR

      * SQR part experiment


Tuesday
NapABC

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

• SQR

      * SQR part experiment

Wednesday
NapABC

      * acquire many colony on the plate
-pick 8 colony on each plate(Cm1~Cm8)
* Colony PCR
Forward primer: CmR gene-R-SpeI
Reverse primer: NapABC-RP
Conclusion: Cm1~ Cm 4. Cm6. Cm7 positive

• SQR

      * SQR part experiment

Thursday
NapABC

      * Enzyme check
MS-pSB1C3-RBS-NapABC
Enzyme: SacI-HF (1307+2406)
Conclusion: positive

• SQR

      * SQR part experiment


Friday
NapABC

      * Send for sequencing
Conclusion: positive

• SQR

      * SQR part experiment


Saturday
SQR

      * SQR part experiment



Week 11 (9/9~9/15)


Sunday
SQR

      * The effect of sodium sulfide on Synechococcus SP. PCC 7942 growth rate
* In this experiment, we aimed to examine whether the SQR Synechococcus SP. PCC 7942 expressed is functional. Synechococcus SP. PCC 7942 with SQR and wild type was cultivated in BG-11 medium (Allen M.M. 1968) on the same rotary shaker described in the first experiment. Initial cell concentration is fixed to an OD730 of 0.1 in the medium. 2.5mM of sodium sulfide is added into the experimental group, and DCMU is diluted from 1μM to 0.5μM.


Monday
NirN-RBS-NirS-RBS-NapABC

* Sub clone NirN-RBS-NirS gene from pSB1C3 and NapABC gene from MS-pSB1C3 into Ptrc-kan plasmid
* Ptrc-kan plasmid cut with EcoRI, XbaI
* MF-pSB1C3 -DsrI-BamHI-DsrII gene Cut with MfeI, SpeI
*(smart trick: MfeI and EcoRI form scar, SbfI and SpeI form scar, to prevent endogenous EcoRI and PstI site)

• SQR

      * SQR part experiment
* DCMU concentration and cell growth
From the last experiment, we found it necessary to perform another independent experiment to find the optimal DCMU concentration of Synechococcus SP. PCC 7942. In addition, the initial cell concentration was adjusted to an OD730 of 0.2 in BG-11 medium. DCMU is diluted from 1μM to 0.1251μM. Except of cell concentration, all condition remained the same for both sqr and wild type strain of Synechococcus SP. PCC 7942.


Tuesday
SQR

      * SQR part experiment


Wednesday
SQR

      * SQR part experiment

Thursday
SQR

      * SQR part experiment

Friday

• SQR

      * SQR part experiment
Saturday

• SQR

      * SQR part experiment