Difference between revisions of "Team:SPSingapore/Notebook-Week-8"

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<span class = "black"> ESA Quorum Sensing</span>
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<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Adrian &#9728;</font>
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Confirmation of gfp plasmid
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RE digest of gfp plasmid with EcoRI/PStI
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<tr><td> EcoRI </td><td> 2ul </td></tr>
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<tr><td> PstI </td><td> 2ul </td></tr>
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<tr><td> Buffer </td><td> 4ul </td></tr>
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<tr><td> DNA </td><td> 10ul </td></tr>
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<tr><td> H2O </td><td> 22ul </td></tr>
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<tr><td> Total </td><td> 40ul </td></tr>
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Ran gel, band size was correct
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<br> gel extraction -> 101ng/ul
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Master plates
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<br> RNG - 4 colonies
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<br> esaRGFP- 6 colonies
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Revision as of 18:47, 17 September 2015


Research Notebook

Week 8 (12/7 - 18/7)

▪ July 13

Anaerobic Promoter    ☀ Yi Han & Yun Ting ☀

Ligation for FNRgfp (4X)
200ng of gfp plasmid (4.36ul)
297.5 ng insert (7:1) 10.3
1ul 10Buffer (53.34)
4ul T4 ligase

Trial run of anaerobe chamber
open sachet to decrease O2 at 3.30pm
takes 2.5h to activate
streak plate of P. putida a strict aerobe and E. coli, facultative aerobe in chamber at 30deg C to grow O/N
P. putida is an obligate aerobe and if chamber works ,it will not grow
E. coli should grow in both conditions
controls grown outside chamber


▪ July 14

Anaerobic Promoter    Invasin + Listerolysin
☀ Yi Han ☀

No colonies for FNRgfp plasmid
Inv Plasmid Verification
buffer 1
EcoRI 0.5
in plasmid 0.5
dH2O 8
Total 10ul


Gel extraction RE digest of EsaR-GFP plasmid
LigationControl
Vector (45ng) 1 1
insert 1 1
Buffer 1 1
dH2O 6 7
ligase 1 1


Transformation of FNRgfp
Anaerobe jar
E. coli grew in both conditions
P putida-> some growth in anaerobe chamber
Proper streak plate in aerobic conditions
Packet runs out after 16 hours


▪ July 15

Anaerobic Promoter    ☀ Yi Han ☀

FNRgfp-> no colonies after transformation
religation (4X reaction)
200ng of gfp plasmid (4.4ul)
2125ng of insert (8ul)
4ul T4 ligase
55.6 H2O


Anaerobic Promoter    ☀ Yi Han & Chi Yan ☀

Transformation of FNR gfp into 20ul of BL21


▪ July 16

Anaerobic Promoter    ☀ Yi Han ☀

No colonies grew
Ran a gel, FNRgfp pcr, gfp pcr, fnr, 100bp ladder
▶ Seems to be band shift.


▪ July 17

Anaerobic Promoter    ☀ Chi Yan ☀

RE digest of FNR-GFP fusion PCR with EcoRI and PstI
EcoRI 1
PstI 1
Buffer 5
FNR-GFP from 12/7 in RIP box 1ug -> 2.5ul
H2O 40.5
Total 50
▶ RE for 2 hours at 37 degrees
▶ Direct purification using Promega kit

Overnight ligation (4X reaction)
200ng of gfp plasmid (4.4ul)
400ng of insert (5:1) (4ul)
4ul T4 ligase
8ul T4 ligase buffer
44.1 H2O


ESA Quorum Sensing    ☀ Adrian ☀

Ligation of esaRBS-GFP (EcoRI/PstI) and RNG-GFP (EcoRI/PstI) into pSB1A2 (EcoRI/PstI)
10x T4 DNA ligase Buffer 1ul
Vector 1ul
Insert 5ul
H2O 10ul
T4 DNA ligase 1ul
▶ Reactions were incubated for 30min at room temperature

Transformation
  pSB1A2 esaRBSGFP
  pSB1A2 RNG GFP
  Control cut pSB1A2


Anaerobic Promoter    ☀ Yi Han ☀

For plates on LB alone there was a lawn
For pUC19, the transformation worked, and separated colonies were produced
No colonies were produced for the FNR-gfp plasmid


▪ July 18

Anaerobic Promoter    ☀ Xin Yi ☀

Transformation of FNRGFP ligated product into BL21 (from Chi Yan 17/7)
   1. 3ul pUC19 control +10ul BL21
   2. 20ul Ligation product + 20ul BL21
   3. 5ul Ligation product + 20ul BL21

Plated on LB+amp plates
   40ul of transformed bacteria in SOC media on LB+amp
   20ul of transformed bacteria in SOC media on LB alone


ESA Quorum Sensing    ☀ Adrian ☀

Confirmation of gfp plasmid
RE digest of gfp plasmid with EcoRI/PStI
EcoRI 2ul
PstI 2ul
Buffer 4ul
DNA 10ul
H2O 22ul
Total 40ul


Ran gel, band size was correct
gel extraction -> 101ng/ul

Master plates
RNG - 4 colonies
esaRGFP- 6 colonies