LB medium (liquid)300ml×2 & (solid)300ml×2 & ddH2O×1
HCZ&JKY
Solution Configuration
0.1M CaCl2×1 & 0.1M CaCl2+15% glycerol×1
In No.8 4℃ refrigerator
Senior Fellow Apprentice
Solution Configuration
100mg/ml Amp×6
In No.1 -20℃ refrigerator
HCZ&JKY
Single colony & flasks culture
Nissle×6 & DH10B×1
Begin at 21:00(BJT), End at 9:00(BJT). Storage in 4℃
ZDH
PCR
bb1 PUC57
Fail（The PCR machine clicked the Not heat cover key, and the liquid in the PCR tube was steamed dry.）
ZDH
Receiving & preserving bacteria
DH5α×4 & W3110×2
In No.8 4℃ refrigerator
JKY
Genome extraction
DH5α×3 & W3110×3
In No.1 -80℃ refrigerator
Electrophoretic Verification
DH5α & W3110 (genome)
Image Storage "iGEM 2019 3.13" Folder.
PCR & Electrophoretic Verification
pUC57 backbone 1
Success
Solution Configuration
50mg/ml Kana×10
In No.1 -20℃ refrigerator
YYJ&WYH
PCR
(2000 and fimH two tubes each) 50 liter system
Inoculated-pathogen
pUC57-kan×2, Shake bacterium overnight
Dissolved PSB plasmid
In No.8 4℃ refrigerator
CYY
Competent cells preparation（Nissle）
In -80℃ refrigerator
GYC&ZDH
Electrophoresis
fimH×2，bb-2×2，lacz×2
Fail（The white shell is not compacted when mixing.）
ZDH
Transmission
Nissle competence was transferred to pUC57 plasmid to verify the characteristics of the covert plasmid.
PUC will not be lost and single colonies will grow.
HJY
Transmission
The amplification of DH10B into pSB3C5 failed. Preliminary analysis indicated that the quality of DH10B was too poor or that some problems occurred during the transformation process resulted in the death of competent cells.
JKY&YYJ&HCZ
PCR
fimH×2；bb-2(Using plasmid as template); 1500&2000; lacZ×2
Transmission
/
ZDH
Rubber Recovery
bb-2 1500&2000 pcr
JKY&HCZ
Transmission
bb-2 1500&2000
Failure of validation may be due to primer problems or BB1 recovered before ZDH.
HCZ
Store lacZ parts and ptetR in Kit Plate 7 in the -20℃ refrigerator.
ZDH
Flasks culture
Get DH10B from the-80℃ refrigerator
Inoculate three test tubes and shake them for the next day.
Inoculated nissle single colony shake
YYJ
Preparation of competent bacteria
Type: DH10B
ZDH
Transmission
The plasmid pSB3C5 was transferred to DH10B. (It was found that it did not grow out at 3.18.)
ZDH&YYJ
PCR & Gel extraction
Dilution 1500 and 2000 times (bb-1).
ZDH
Transmission
Discarding the product recovered from yesterday's glue and re-preparing two Bb-1 tubes.
The transformation failed to validate the two parts required by JKY. Preliminary analysis showed that the concentration of plasmid given by iGEM was too low.
JKY&YYJ
Gradient PCR
pCU57-lacZ
PCR
pSB3C5
JKY
Gene Connection
lacZ and pUC57-KanR-kan pro
Transmission
Transform into DH10B, coating plate.
Fail, didn't grow, while the control group grew.
Electrophoretic Verification
lacZ、pUC57-KanR-kan pro、Original pUC57-KanR
ZDH
PCR of bb-2. Fail, gel image saved, approximately 1.5kb, should be around 3kb
ZDH&GYC
PCR of pSB3C5-vector, tet operator and fimH for one-step cloning, gel recycled
ZDH
One-step cloning of 3 parts mentioned above
LJH
One-step cloning of JKY's pUC57-kan pro
HCZ
PCR of pUC57-KanR-kan pro-BB, gel recycled
JKY
one-step cloning of lacZ and pUC57-KanR-kan pro-BB
PCR of lacZ from DH5α、w3110、DH10B 、fimH,gel recycled anddetecting concentrations, lacZ sequencing
ZDH
gel recycled, one gel image saved, pcr
CYY
PCR of pUC57bb,gel recycled
JKY
PCR of pGH-bb, lacZ; gel recycled; connected; transformed
JKY
Pick single colony, shake it over night
HCZ
1）Take 8 tubes of bacteria that contain pGH-lacZ from the incubator shakers. 2) ⑤、⑥tubes are wrongly added to bacteria retaining tubes①②、③④，which waste ⑤、⑥tubes. 3)Plasmid extraction of ①②③④⑦⑧ and the protucts were in the -4℃ refrigerator.
JKY
pGH-lacZ was tested by HindIII, SspI, EcoRI, BglI, and the result is not satisfactory, and we were told that the backbone we use is not for expressing protein.
JKY
PCR p15A-Amp-BB and lacZ
DNA extraction of ZDH's 8 tube of p15A-kanR-fimH
YYJ
p15A-BB and lacZ from (DH5alpha) gel recycled, connected, and transformed into DH10B
Incubate DH10B with p15A-lacZ(#3) in 2test tubes overnight
Incubate nissle with p15A-fimH in 4 test tubes overnight
Sequencing primers of pUC-GFP sent
ZDH
Cryopreservation of Nissle-p15A-fimH-knaR strains: 14A-3-D1~8, note that DH10B-p15A-fimH-knaR strains are at 14A-3-C1~3.
Bacteria solution PCR of nissle-p15A-fimH-knaR to verify the existence of fimH in nissle. All lanes are bright, including negative control, suggesting nissle has fimH in its genome.
HCZ
Incubate cryopreserved DH10B with p15A-lacZ in 6 tubes at 9:10 am. BUT Glycerin was added by mistake!
JKY&YYJ
We aimed to Incubate cryopreserved DH10B with p15A-lacZ for minprep practice, but what is worth reflecting on is that we added wrong resistance(in fact it is AmpR, but we added KanR). BTW, HCZ forgot to write "AmpR" on the Bacteria retaining tube, JKY forgot to check the sanpgene before adding resistance, and YYJ forgot that once she wrote Amp on the petri dish. In a workplace, for every accident that causes a major injury, there are 29 accidents that cause minor injuries and 300 accidents that cause no injuries
from
JKY
Transformation of pKD46(contains RED Homologous Recombination System) into DH10B(for amplification), incubate in 30℃ for 12-16h
ZDH&YYJ
Miniprep of p15A-Kana-fimH ×6
JKY
Inoculation of wild Nissle(without any resistance) from bacteria retaining tube ×2, pick away at 9:00 tommorrow
HJY
MlacI PCR test2 succeeded
JKY
Miniprep of pKD46-Amp-30℃×4
Test pKD46-Amp-30℃ by SspI
HCZ
Store 4 tube of pKD46-Amp-30℃
JKY
Test nissle by ONPG with different propotion
JKY&HCZ
Transform p15A-Amp-lacZ into Nissle
Measure the growth curve of Nissle for 8h
JKY
Test Nissle by ONPG with different propotion again on ELIASA
Incubate Nissle with p15A-Amp-lacZ in 37℃
YYJ
Miniprep of ZDH's pUC-GFP-AmpR-KanR ×8
ZDH
Bacteria solution PCR of nissle-p15A-lacZ1&2, confirming plasmid to be right
Transmission of p15A-lacZ-AmpR into Nissle, incubate on solid LB at 37℃
Incubate nissle-p15A-lacZ1&2(supposed to be contaminated) on solid LB at 37℃ overnight to purify them.
JKY
The prsentation of MRS(Anaerobic medium)
TPre-experiment of plate count methond(E.coli) and tube count method(anaerobion)
Incubate nissle-p15A-lacZ1&2(supposed to be contaminated) on solid LB at 37℃ overnight(because stupid Zhu added 150ul bacteria solution last time, and the plates were too ugly to pick a single colony)
YYJ
ONPG of Nissle in different lactose concentration
JKY
Presentation of LB(solid)×4
Pre-experiment of plate count methond(E.coli) and tube count method(anaerobion) again with better degree of dilution
YYJ
ONPG for Nissle（lactose concentration0.2-2.0mg/ml）,DH10B（lactose concentration0.2-0.6） Nissle-p15A-Amp-lacZ(0.2-1.0) in different lactose concentration（bacterium concentration 20%）
incubate Nissle-p15A-Amp-lacZ①、②
pick 1 single colonies of Nissle-P15A-lacZ, shake it overnight
ZDH
Ligation of pUC-J23106-luxI-B0015-AmpR-knaR and transmitted it into DH10B, incubate in solid LB at 37℃ overnight
Ligation of p15A-aafA-knaR-GFP and transmitted it into DH10B, incubate in solid LB at 37℃ overnight
Ligation of p15A-agg3A-knaR-GFP and transmitted it into DH10B, incubate in solid LB at 37℃ overnight
Incubate S. cerevisiae in YPD(l) at 30℃ for 24hrs
HJY&CYY
OE PCR of J23106-luxI-B0015
ZDH
Incubate p15A-aafA-knaR-GFP in liquid LB overnight (select 8 monoclone)
Incubate p15A-agg3A-knaR-GFP in liquid LB overnight (select 7 monoclone)
Incubate pUC-J23106-luxI-B0015-AmpR-KanR in liquid LB overnight (select 1 monoclone ) note that there's only one monoclone on 2plates together, I've got to make sure its not because double antibiotics were added to the petri dish. So I've put it into incubator overnight to see if new monoclones come up tomorrow morning.
ZDH
Enzyme digest of p15A-aafA-knaR-GFP and p15A-agg3A-knaR-GFP ,save fA#4&5, 3A#5, 6&8
ZDH
Enzyme digesting fA#4&5, 3A#5, 6&8, luxI, proving only 3A#5&6 are correct
ZDH
Incubate agg3A 5-1, 6-1, DH10B, nissle, nissle-fimH in test tubes overnight
Select monoclone of DH10B and luxR and incubate in test tube
Gel recycle B0015
Transform pUC-GFP-kanR-AmpR into DH10B to prove reliability of competent DH10B
ZDH
Incubate agg3A 5-1, 6-1, DH10B, nissle, nissle-fimH in Erlenmeyer.
Plasmid extraction of agg3A 5-1, 6-1and sent them for sequencing
CYY
PCR of LuxI, LuxR, B0015, backbone
CYY
Transmission of J23106-RBS-LuxR-B0015-bb(106) into DH10B, plated on LB agarplate with Kan and Amp
Transmission of J23106-RBS-LuxR-B0015-bb(sec) into DH10B, plated on LB agarplate with Kan and Amp
CYY
CE ligation reaction of J23106-RBS-LuxI-B0015-bb(106) and J23106-RBS-LuxI-B0015-bb(sec)
Transmission of J23106-RBS-LuxI-B0015-bb(106) and J23106-RBS-LuxI-B0015-bb(sec) into DH10B, plated on LB agarplate with Kan and Amp
Failed
HCZ
1)No colonies were found in the LB medium with 10-7 and 10-8 yogurt diluent after 18hours. About 10 colonies are in the MAC medium with nissle after 18hours. About 5 colonies are in the MAC medium with yogurt diluent after 18hours. 2)measuring growth curve of nissle in 3 groups for 12 hours
JKY
Try direct microscopic count. PCR of single colony on MAC plate, all have fimH
CYY
CE ligation reaction of J23106-RBS-LuxI-B0015-bb(106) and J23106-RBS-LuxI-B0015-bb(sec)
HJY
12 luxR-B0015 colonies miniprep，enzyme digestion test，all seems to be right
ZDH
Incubate 20ul yogurt on MRS(s)(1:100) at 37 ℃ overnight
Incubate 30ul nissle (cryopreserved) on LB(s)at 37℃ overnight
Bacteria PCR of nissle, DH10B to check the nissle-specific primer
By HCZ
PCR, p15A-kanR-ptetR-agg3A-GFP
Fragment
Primer Used
Tm and cycle number
Length
GFP-p15A-kanR-ptetR-
gfp-ptetR-F
60 35cycle
3212
p15A-Vector-R
By ZDH
Transmission to prove viability of competent cell (chemical)
Cell name
Plasmid name
Incubate temperature
Incubate time
result
DB3.1
pUC-GFP-AmpR-kanR
37
14h
Good
DH10B
pUC-GFP-AmpR-kanR
37
14h
Good
Nissle
pUC-GFP-AmpR-kanR
37
14h
No clone
Inoculate bacteria in test tubes
Cell name
Plasmid name
Antibiotics
Number of test tubes
Incubate temperature
Incubate time
Result
DB3.1
pBR322-tetR-tetO-AmpR-cyg
Amp
2
37
14h
Good
pBR322-tetR-tetO-AmpR-cyg
Cl
2
37
14h
Good
By ZDH
Plasmid extraction
Plasmid name
Concentration
260/280
Enzyme digestion
Result
pBR322-tetR-tetO-AmpR-cyg
/
/
BcuI+Eco105I
good
P15A-tetR-tetO-cmR-cyg
/
/
Xbal
good
By YYJ
CE ligation reaction of p15A-ptetR-GFP-KanR
Fragment1
Fragment2
Length
Result
p15A-ptetR-GFP-KanR
p15A-ptetR-GFP-KanR
/
Good
Transmission of p15A-ptetR-GFP-KanR into DH10B
Cell Name
Plasmid Name
Incubate Temperature
Incubate Time
Result
DH10B
p15A-ptetR-GFP-KanR
37℃
14h
Good
By CYY
PCR of luxpr-GFP, PCR of p15A backbone(AmpR)
Fragment
Primer Used
Tm and cycle number
Length
Concentration(after gel purification)
260/280
Luxpr-GFP
Luxprfin-F
58 35cycle
1024
/
/
Luxprfin-R
/
/
/
/
P15A backbone
Luxprfin-F
58 35cycle
1936
/
/
Luxprfin-R
/
/
/
/
CE ligation of pUC-luxI-luxR-AmpR-KanR
Fragment1
Fragment2
Length
Result
pUC-luxR-AmpR
pUC-luxI-KanR
5114
/
CE ligation of pUC-luxI-luxR-AmpR-KanR
Fragment1
Fragment2
Length
Result
luxpr-GEP
P15A-AmpR backbone
2920
success
Transmission of pUC-luxI-luxR-AmpR-KanR into DH10B
Cell name
Plasmid name
Incubate Temperature
Incubate time
Result
DH10B
pUC-luxI-luxR-AmpR-KanR
37℃
/
/
Luxpr-E8.9
luxI:
luxR:14-2-3-d8.9
luxIR:H2.3.4
luxIR-ampR:89, purple, five tubes
Send sequencing of p15A-kanR-ptetR-GFP(plasmid1), Gel Extraction
By YYJ
CE ligation reaction of p15A-KanR-tetR-kanR-ptetR-GFP (plasmid2.0), Transmission of p15A-KanR-tetR-kanR-ptetR-GFP (plasmid2.0）
Cell name
Plasmid name
Incubate Temperature
Incubate time
Result
DH10B
p15A-KanR-tetR-kanR-ptetR-GFP (plasmid2.0)
37℃
18h
/
By ZDH
PCR
Template
Primer
Length(kb)
Gel recycle concentration (ng/ul)
P15A-kanR-AmpR-J23106-luxR-B0015
J23106-F/R
0.2
/
P15A-kanR-ptetR-GFP-B0014
bb-p15A-ptetR-GFP-F/R
3.0
/
pBR322-amp-tetR-tetO-hyg-ccdB
tetR-F/R
0.7
/
Recombination and transformation
Bacteria
Plasmid
Antibiotics (concentration)
Temperature
Time
Result
DH10B
J23106-F/R+bb-p15A-ptetR-GFP-F/R+tetR-F/R
kanR
37℃
18h
good
By ZDH
Select colonies and incubate
Bacteria
Plasmid
Number of colonies selected
Temperature
Time
Result
DH10B
P15A-kanR-J23106-tetR-ptetR-GFP (#1-#6)
6
37℃
16h
good
By ZDH
Plasmid extraction
Plasmid name
Concentration
260/280
Enzyme digestion
Result
Sequencing
notes
P15A-kanR-J23106-tetR-ptetR-GFP (#1-#6)
/
/
NdeI+XhoI
#1, #3, #5 confirmed
#1 confirmed
Cryopreserved #1, #3, #5
By ZDH
Plasmid extraction
Plasmid name
Concentration
260/280
Enzyme digestion
Result
notes
P15A-kanR-J23106-tetR-ptetR-GFP
/
/
/
good
For PCR template
By HCZ
PCR
Template
Primer
Length(kb)
Gel recycle concentration (ng/ul)
P15A-kanR-J23106-tetR-ptetR-GFP
J23100-F+BT-R
0.7
46.5
P15A-kanR-J23106-tetR-ptetR-GFP
J23100-R+BT-F
3.0
22.1
P15A-kanR-J23106-tetR-ptetR-GFP
J23118-F+BT-R
0.7
55.4
P15A-kanR-J23106-tetR-ptetR-GFP
J23118-R+BT-F
3.0
15.8
P15A-kanR-J23106-tetR-ptetR-GFP
J23116-F+BT-R
0.7
24.5
P15A-kanR-J23106-tetR-ptetR-GFP
J23116-R+BT-F
3.0
22.1
P15A-kanR-J23106-tetR-ptetR-GFP
J23112-F+BT-R
0.7
27.5
P15A-kanR-J23106-tetR-ptetR-GFP
J23112-R+BT-F
3.0
21.8
P15A-kanR-J23106-tetR-ptetR-GFP
J23110-F+BT-R
0.7
59.3
P15A-kanR-J23106-tetR-ptetR-GFP
J23110-R+BT-F
3.0
22.1
P15A-kanR-J23106-tetR-ptetR-GFP
J23104-F+BT-R
0.7
77.7
P15A-kanR-J23106-tetR-ptetR-GFP
J23104-R+BT-F
3.0
30.4
By ZDH
Recombination and transformation
Bacteria
Plasmid
Antibiotics (concentration)
Temperature
Time
Result
DH10B
100-F/BT-R+100-R/BT-F
kanR (1x)
37℃
16h
good
DH10B
104-F/BT-R+104-R/BT-F
kanR (1x)
37℃
16h
good
DH10B
110-F/BT-R+110-R/BT-F
kanR (1x)
37℃
16h
good
DH10B
112-F/BT-R+112-R/BT-F
kanR (1x)
37℃
16h
good
DH10B
116-F/BT-R+116-R/BT-F
kanR (1x)
37℃
16h
No clone
DH10B
118-F/BT-R+118-R/BT-F
kanR (1x)
37℃
16h
good
By ZDH
Select colonies and incubate
Bacteria
Plasmid
Number of colonies selected
Temperature
Time
Result
DH10B
P15A-kanR-J23100-tetR-ptetR-GFP
4
37℃
14h
good
DH10B
P15A-kanR-J23118-tetR-ptetR-GFP
4
37℃
14h
good
DH10B
P15A-kanR-J23112-tetR-ptetR-GFP
4
37℃
14h
good
DH10B
P15A-kanR-J23110-tetR-ptetR-GFP
2
37℃
14h
good
DH10B
P15A-kanR-J23104-tetR-ptetR-GFP
4
37℃
14h
good
By ZDH
Plasmid extraction
Plasmid name
Concentration
260/280
Enzyme digestion
Result
Sequencing
notes
P15A-kanR-J23100-tetR-ptetR-GFP(No.1-No.4)
/
/
NdeI+XhoI
Confirmed No.2, No.3, No.4
Cryopreserved No.2
/
P15A-kanR-J23118-tetR-ptetR-GFP(No.1)
/
/
NdeI+XhoI
Confirmed
Cryopreserved
/
P15A-kanR-J23112-tetR-ptetR-GFP(No.1)
/
/
NdeI+XhoI
Confirmed
Cryopreserved
/
P15A-kanR-J23110-tetR-ptetR-GFP(No.1)
/
/
NdeI+XhoI
Confirmed
Cryopreserved
/
P15A-kanR-J23104-tetR-ptetR-GFP(No.1)
/
/
NdeI+XhoI
Confirmed
Cryopreserved
/
That incubated bacteria shows green fluorescence proves there exists defects in plasmid design.. We assume that the bacterial terminator doesn't give a complete stop to tetR expressed by J23 promoters, and thus we decided to reconstruct our testing plasmid.
Notebook