Team:Stockholm/15 October 2010
From 2010.igem.org
(New page: {{Stockholm/Top2}} ==Andreas==) |
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+ | ==Nina== | ||
+ | |||
+ | ===Continuation of protein purification=== | ||
+ | |||
+ | ====Washing==== | ||
+ | |||
+ | I washed the column with 10 X volumes of wash buffer. | ||
+ | |||
+ | The Ni-resin (in the form of pellet) is 10 ml, therefore 10 * 10ml Ni-NTA = 100 ml wash buffer. | ||
+ | |||
+ | The imidazole is 2 M. | ||
+ | |||
+ | 2M * Volume = 100 * 30 *10^-3 M Volume imidaziole that needs to be added in the wash 100 ml is 1.5 ml | ||
+ | |||
+ | Washing: | ||
+ | |||
+ | [[Image:Aq14.jpg|300px]] | ||
+ | |||
+ | ====Elution==== | ||
+ | |||
+ | I eluted the column with 5 X volumes of elution buffer. | ||
+ | |||
+ | The Ni-resin (in the form of pellet) is 10 ml, therefore 5 * 10ml Ni-NTA = 50 ml elution buffer. | ||
+ | |||
+ | *20 ml 40 min on shake 4°C | ||
+ | *10 ml 30 min on shake 4°C | ||
+ | *10 ml 30 min on shake 4°C | ||
+ | *10 ml 30 min on shake 4°C | ||
+ | |||
+ | I saved these samples in the cold room 4°C for tomorrow. | ||
+ | |||
+ | ===Agarose gel verification=== | ||
+ | |||
+ | I ran the PCR products on an 1 % agarose gel 80 V to check if I had any inserts. | ||
+ | |||
+ | Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp | ||
+ | |||
+ | [[Image:Aq12.jpg|150px]] | ||
+ | |||
+ | Arrangement on gel: | ||
+ | |||
+ | [[Image:Aq13.jpg]] | ||
+ | |||
+ | Gels: | ||
+ | |||
+ | [[Image:Aq15.jpg|250px]] | ||
+ | |||
+ | Unfortunately non of the bands show that there has been an insert, it might be that the transformation into BL21 cells was not a good idea since they are not cloning cells, but we are short on time and I tried to make a short cut. It might have failed this time. I will run a new screen with 16 colonies/dish, it that won't work then this cloning has not worked out well. | ||
+ | |||
==Andreas== | ==Andreas== | ||
+ | ===Growth curve assay=== | ||
+ | ====Setup==== | ||
+ | Inoculated new flasks from ON cultures set 14/10: | ||
+ | *250 ml E-flasks | ||
+ | *25 ml LB + Amp 100 + 1 mM IPTG | ||
+ | *250 μl ON culture | ||
+ | *#BL21 pEX.SOD⋅His | ||
+ | *#BL21 pEX.nTra10⋅SOD⋅His | ||
+ | *#BL21 pEX.nTAT⋅SOD⋅His | ||
+ | *#BL21 pEX.nLMWP⋅SOD⋅His | ||
+ | *37 °C, 225 rpm | ||
+ | |||
+ | OD<sub>590</sub> measurements made every 30 min with appropriate dilution in fresh LB. | ||
+ | |||
+ | ====OD<sub>590</sub> measurements==== | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | | | ||
+ | !width="50"|0 min | ||
+ | !width="50"|30 min | ||
+ | !width="50"|60 min | ||
+ | !width="50"|90 min | ||
+ | !width="50"|120 min | ||
+ | !width="50"|150 min | ||
+ | !width="50"|180 min | ||
+ | !width="50"|210 min | ||
+ | !width="50"|240 min | ||
+ | !width="50"|270 min | ||
+ | !width="50"|300 min | ||
+ | !width="50"|330 min | ||
+ | !width="50"|360 min | ||
+ | !width="50"|390 min | ||
+ | |- | ||
+ | |'''pEX.SOD⋅His''' | ||
+ | |align="center"|0.035 | ||
+ | |align="center"|0.047 | ||
+ | |align="center"|0.090 | ||
+ | |align="center"|0.180 | ||
+ | |align="center"|0.195 | ||
+ | |align="center"|0.176 | ||
+ | |align="center"|0.251 | ||
+ | |align="center"|0.177 | ||
+ | |align="center"|0.239 | ||
+ | |align="center"|0.140 | ||
+ | |align="center"|0.164 | ||
+ | |align="center"|0.205 | ||
+ | |align="center"|0.196 | ||
+ | |align="center"|0.193 | ||
+ | |- | ||
+ | |'''pEX.nTra10⋅SOD⋅His''' | ||
+ | |align="center"|0.037 | ||
+ | |align="center"|0.050 | ||
+ | |align="center"|0.092 | ||
+ | |align="center"|0.163 | ||
+ | |align="center"|0.156 | ||
+ | |align="center"|0.132 | ||
+ | |align="center"|0.175 | ||
+ | |align="center"|0.130 | ||
+ | |align="center"|0.164 | ||
+ | |align="center"|0.116 | ||
+ | |align="center"|0.155 | ||
+ | |align="center"|0.167 | ||
+ | |align="center"|0.154 | ||
+ | |align="center"|0.168 | ||
+ | |- | ||
+ | |'''pEX.nTAT⋅SOD⋅His''' | ||
+ | |align="center"|0.033 | ||
+ | |align="center"|0.046 | ||
+ | |align="center"|0.090 | ||
+ | |align="center"|0.170 | ||
+ | |align="center"|0.177 | ||
+ | |align="center"|0.161 | ||
+ | |align="center"|0.225 | ||
+ | |align="center"|0.153 | ||
+ | |align="center"|0.225 | ||
+ | |align="center"|<font style="color:red">0.045</font> | ||
+ | |align="center"|0.170 | ||
+ | |align="center"|0.162 | ||
+ | |align="center"|0.193 | ||
+ | |align="center"|0.200 | ||
+ | |- | ||
+ | |'''pEX.nLMWP⋅SOD⋅His''' | ||
+ | |align="center"|0.031 | ||
+ | |align="center"|0.042 | ||
+ | |align="center"|0.86 | ||
+ | |align="center"|0.174 | ||
+ | |align="center"|0.192 | ||
+ | |align="center"|0.178 | ||
+ | |align="center"|0.237 | ||
+ | |align="center"|0.170 | ||
+ | |align="center"|0.242 | ||
+ | |align="center"|0.137 | ||
+ | |align="center"|0.180 | ||
+ | |align="center"|0.185 | ||
+ | |align="center"|0.200 | ||
+ | |align="center"|0.200 | ||
+ | |- | ||
+ | |'''Dilution factor''' | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|2 | ||
+ | |align="center"|4 | ||
+ | |align="center"|5 | ||
+ | |align="center"|10 | ||
+ | |align="center"|10 | ||
+ | |align="center"|20 | ||
+ | |align="center"|20 | ||
+ | |align="center"|20 | ||
+ | |align="center"|20 | ||
+ | |align="center"|20 | ||
+ | |} | ||
+ | |||
+ | [[image:Growth_assay_plot_15oct.png|200px|thumb|right|'''Line chart of plotted OD(590) measurements from growth assay.''']] | ||
+ | |||
+ | ====Results==== | ||
+ | Results indicate no significant difference in growth between different cultures. Slightly lower curve for the nTra10-expressing clone, which is somewhat interesting, since a similar progress was seen in the canceled experiment from 14/10. At least one more experiment will be run next week, using new BL21 clones; this will show whether this is just a coincidence or not. | ||
+ | |||
+ | |||
+ | |||
+ | == Mimmi == | ||
+ | |||
+ | === clone non-CPP operon === | ||
+ | |||
+ | ==== colony PCR ==== | ||
+ | |||
+ | {| | ||
+ | ! mix | ||
+ | | | ||
+ | | x16 | ||
+ | | rowspan="5" width="150" | | ||
+ | ! primers | ||
+ | | rowspan="5" width="150" | | ||
+ | ! samples | ||
+ | |- | ||
+ | | mastermix | ||
+ | | 24.5 | ||
+ | | | ||
+ | | pSB_VF2 | ||
+ | | pSB1C3.SOD.RBS.yCCS 1-5 | ||
+ | |- | ||
+ | | DNA | ||
+ | | 0.5 | ||
+ | | | ||
+ | | pSB_VR | ||
+ | | pSB1C3.SOD.his.RBS.yCCS 1-5 | ||
+ | |- | ||
+ | | align="right" | tot | ||
+ | | 25µl | ||
+ | | | ||
+ | | rowspan="2" | | ||
+ | | pSB1C3.his.SOD.RBS.yCCS 1-5 | ||
+ | |- | ||
+ | | colspan="3" | | ||
+ | | positive control (pSB1C3.LMWP.SOD.his) | ||
+ | |} | ||
+ | |||
+ | ==== Gel ==== | ||
+ | |||
+ | [[Image:2010-10-15 pC.S yC.jpg|200px|thumb|left|]] | ||
+ | {| | ||
+ | ! well | ||
+ | ! sample | ||
+ | ! well | ||
+ | ! sample | ||
+ | |- | ||
+ | | 1 | ||
+ | | ladder | ||
+ | | 12 | ||
+ | | ladder | ||
+ | |- | ||
+ | | 2 | ||
+ | | pC.S_yC 1 | ||
+ | | 13 | ||
+ | | pC.hS_yC 1 | ||
+ | |- | ||
+ | | 3 | ||
+ | | pC.S_yC 2 | ||
+ | | 14 | ||
+ | | pC.hS_yC 2 | ||
+ | |- | ||
+ | | 4 | ||
+ | | pC.S_yC 3 | ||
+ | | 15 | ||
+ | | pC.hS_yC 3 | ||
+ | |- | ||
+ | | 5 | ||
+ | | pC.S_yC 4 | ||
+ | | 16 | ||
+ | | pC.hS_yC 4 | ||
+ | |- | ||
+ | | 6 | ||
+ | | pC.S_yC 5 | ||
+ | | 17 | ||
+ | | pC.hS_yC 5 | ||
+ | |- | ||
+ | | 7 | ||
+ | | pC.Sh_yC 1 | ||
+ | | 18 | ||
+ | | pC.L.hS, control | ||
+ | |- | ||
+ | | 8 | ||
+ | | pC.Sh_yC 2 | ||
+ | | rowspan="4" | | ||
+ | | rowspan="4" | | ||
+ | |- | ||
+ | | 9 | ||
+ | | pC.Sh_yC 3 | ||
+ | |- | ||
+ | | 10 | ||
+ | | pC.Sh_yC 4 | ||
+ | |- | ||
+ | | 11 | ||
+ | | pC.Sh_yC 5 | ||
+ | |} | ||
+ | |||
+ | |||
+ | ==== ON culture ==== | ||
+ | |||
+ | 3ml LB Cm<sub>25</sub> + toothpick from colony | ||
+ | |||
+ | SOD.RBS.yCCS 5 | ||
+ | SOD.his.RBS.yCCS 2 | ||
+ | his.SOD.RBA.yCCS 3 | ||
+ | |||
+ | {{Stockholm/Footer}} |
Latest revision as of 11:10, 26 October 2010
Contents |
Nina
Continuation of protein purification
Washing
I washed the column with 10 X volumes of wash buffer.
The Ni-resin (in the form of pellet) is 10 ml, therefore 10 * 10ml Ni-NTA = 100 ml wash buffer.
The imidazole is 2 M.
2M * Volume = 100 * 30 *10^-3 M Volume imidaziole that needs to be added in the wash 100 ml is 1.5 ml
Washing:
Elution
I eluted the column with 5 X volumes of elution buffer.
The Ni-resin (in the form of pellet) is 10 ml, therefore 5 * 10ml Ni-NTA = 50 ml elution buffer.
- 20 ml 40 min on shake 4°C
- 10 ml 30 min on shake 4°C
- 10 ml 30 min on shake 4°C
- 10 ml 30 min on shake 4°C
I saved these samples in the cold room 4°C for tomorrow.
Agarose gel verification
I ran the PCR products on an 1 % agarose gel 80 V to check if I had any inserts.
Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
Arrangement on gel:
Gels:
Unfortunately non of the bands show that there has been an insert, it might be that the transformation into BL21 cells was not a good idea since they are not cloning cells, but we are short on time and I tried to make a short cut. It might have failed this time. I will run a new screen with 16 colonies/dish, it that won't work then this cloning has not worked out well.
Andreas
Growth curve assay
Setup
Inoculated new flasks from ON cultures set 14/10:
- 250 ml E-flasks
- 25 ml LB + Amp 100 + 1 mM IPTG
- 250 μl ON culture
- BL21 pEX.SOD⋅His
- BL21 pEX.nTra10⋅SOD⋅His
- BL21 pEX.nTAT⋅SOD⋅His
- BL21 pEX.nLMWP⋅SOD⋅His
- 37 °C, 225 rpm
OD590 measurements made every 30 min with appropriate dilution in fresh LB.
OD590 measurements
0 min | 30 min | 60 min | 90 min | 120 min | 150 min | 180 min | 210 min | 240 min | 270 min | 300 min | 330 min | 360 min | 390 min | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
pEX.SOD⋅His | 0.035 | 0.047 | 0.090 | 0.180 | 0.195 | 0.176 | 0.251 | 0.177 | 0.239 | 0.140 | 0.164 | 0.205 | 0.196 | 0.193 |
pEX.nTra10⋅SOD⋅His | 0.037 | 0.050 | 0.092 | 0.163 | 0.156 | 0.132 | 0.175 | 0.130 | 0.164 | 0.116 | 0.155 | 0.167 | 0.154 | 0.168 |
pEX.nTAT⋅SOD⋅His | 0.033 | 0.046 | 0.090 | 0.170 | 0.177 | 0.161 | 0.225 | 0.153 | 0.225 | 0.045 | 0.170 | 0.162 | 0.193 | 0.200 |
pEX.nLMWP⋅SOD⋅His | 0.031 | 0.042 | 0.86 | 0.174 | 0.192 | 0.178 | 0.237 | 0.170 | 0.242 | 0.137 | 0.180 | 0.185 | 0.200 | 0.200 |
Dilution factor | 1 | 1 | 1 | 1 | 2 | 4 | 5 | 10 | 10 | 20 | 20 | 20 | 20 | 20 |
Results
Results indicate no significant difference in growth between different cultures. Slightly lower curve for the nTra10-expressing clone, which is somewhat interesting, since a similar progress was seen in the canceled experiment from 14/10. At least one more experiment will be run next week, using new BL21 clones; this will show whether this is just a coincidence or not.
Mimmi
clone non-CPP operon
colony PCR
mix | x16 | primers | samples | |||
---|---|---|---|---|---|---|
mastermix | 24.5 | pSB_VF2 | pSB1C3.SOD.RBS.yCCS 1-5 | |||
DNA | 0.5 | pSB_VR | pSB1C3.SOD.his.RBS.yCCS 1-5 | |||
tot | 25µl | pSB1C3.his.SOD.RBS.yCCS 1-5 | ||||
positive control (pSB1C3.LMWP.SOD.his) |
Gel
well | sample | well | sample |
---|---|---|---|
1 | ladder | 12 | ladder |
2 | pC.S_yC 1 | 13 | pC.hS_yC 1 |
3 | pC.S_yC 2 | 14 | pC.hS_yC 2 |
4 | pC.S_yC 3 | 15 | pC.hS_yC 3 |
5 | pC.S_yC 4 | 16 | pC.hS_yC 4 |
6 | pC.S_yC 5 | 17 | pC.hS_yC 5 |
7 | pC.Sh_yC 1 | 18 | pC.L.hS, control |
8 | pC.Sh_yC 2 | ||
9 | pC.Sh_yC 3 | ||
10 | pC.Sh_yC 4 | ||
11 | pC.Sh_yC 5 |
ON culture
3ml LB Cm25 + toothpick from colony
SOD.RBS.yCCS 5 SOD.his.RBS.yCCS 2 his.SOD.RBA.yCCS 3
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