▪ 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
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▪ 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
| Ligation | Control |
| 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
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▪ 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
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▪ 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
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