Team:TUDelft/19 August 2009

From 2009.igem.org

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='''19 August 2009'''=
='''19 August 2009'''=
 +
===Sriram===
 +
Today I did minipreps of all the biobricks I cultures yesterday. then again I continued with the assembly by digesting all the parts with the SpeI available in our lab. I am not very confident of it since the expiry date shows somewhere in 2008. So I thought let me continue the work properly after discussing with supervisors on friday because Daniel's backup plan biobricks are working very well.
===Tim Weenink===
===Tim Weenink===
After yesterdays horrible discovery that the I-SceI Homing Endonuclease we got from MIT was actually T4 DNA ligase we had to adjust our cloning strategy. The more primitive part without promoter was found to be the correct one, except that is was assembled with part !A instead of p(LacI). So we did that assembly again today. With p(LacI) from the delay team upstream, *T1 (downstream) and backbone pSB1C3 (also from the delay team). Both RbCl chemically comp and EC electrocomp cells were transformed.  
After yesterdays horrible discovery that the I-SceI Homing Endonuclease we got from MIT was actually T4 DNA ligase we had to adjust our cloning strategy. The more primitive part without promoter was found to be the correct one, except that is was assembled with part !A instead of p(LacI). So we did that assembly again today. With p(LacI) from the delay team upstream, *T1 (downstream) and backbone pSB1C3 (also from the delay team). Both RbCl chemically comp and EC electrocomp cells were transformed.  
 +
 +
 +
===Saeed===
 +
[[Image:Saeed19082009wiki.jpg|thumb|center|250px]]
 +
 +
{| border="1" align="center"
 +
| lane || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  ||
 +
|- align="center"
 +
| 2 || !Fr1n upstream  || 1900 || ok, but not needed
 +
|- align="center"
 +
| 3 || !Fr1n downstream || 1900 || ok
 +
|- align="center"
 +
| 4 || destination plasmid || 3000 || ok
 +
|}
 +
 +
!Fr1n downstream will be purified from the gel by gel extraction and used for the new stategy as Tim mentioned before.
 +
 +
===Daniel===
 +
 +
Digestion of biobricks:
 +
 +
[[Image:gel180809.jpg|550px]]
 +
 +
{| border="1" align="center"
 +
| Well || Biobrick || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  ||
 +
|- align="center"
 +
| 2 || C0051 || 750 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 3 || I13507 || 861 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || P0440 || 840 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5 || I13504 || 875 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 6 || J04630 || 857 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 7 || R0051 || 44 || too small
 +
|- align="center"
 +
| 8 || B0015 || 129 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 9 || J13002 || 74 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 10 || pSB1C3 || 2072 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 11 || pSB1AK3 || 3426 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 12 || pSB1A3 || 2157 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 13 || CB ||  ||
 +
|- align="center"
 +
| 14 || CC ||  ||
 +
|- align="center"
 +
| 15 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  ||
 +
|}
 +
 +
Assemblies 1A, 2A, 3A, 4A, 5A and 6A
 +
 +
Transformation and culture in plates and tubes
 +
 +
===Calin===
 +
 +
Good results on the conjugation test. All plates were imaged on the safe imager with the 5 Megapixel camera. Colony counting was done with [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1770926 Clono-Counter].
 +
 +
For the trbK conjugation test:
 +
 +
{| border="1" align="center"
 +
|-
 +
| <b>Set 1</b>
 +
|- align="center"
 +
| Plate ID || Antibiotics || Dilution ||  # Colonies
 +
|- align="center"
 +
| D1tr || TRI || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| D2tr || TRI || 10<sup>-1</sup>|| overgrown
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-trbK_D3tr.jpeg D3tr] || TRI || 10<sup>-2</sup>|| <font color=green><B>4450</b></font>
 +
|- align="center"
 +
| D4tr || TRI || 10<sup>-3</sup>|| 714
 +
|- align="center"
 +
| D5tr || TRI || 10<sup>-4</sup>|| 86
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-trbK_T1tr.jpeg T1tr] || TRI + CAM || 10<sup>0</sup> || <font color=green><B>2</b></font>
 +
|- align="center"
 +
| T2tr || TRI + CAM || 10<sup>-1</sup>|| 0
 +
|- align="center"
 +
| T3tr || TRI + CAM || 10<sup>-2</sup>|| 0
 +
|- align="center"
 +
| T4tr || TRI + CAM || 10<sup>-3</sup>|| 0
 +
|- align="center"
 +
| T5tr || TRI + CAM || 10<sup>-4</sup>|| 0
 +
|- align="center"
 +
| R1tr || CAM || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| R2tr || CAM || 10<sup>-1</sup>|| 8636
 +
|}
 +
 +
<br><br>
 +
{| border="1" align="center"
 +
|-
 +
| <b>Set 2</b>
 +
|- align="center"
 +
| Plate ID || Antibiotics || Dilution ||  # Colonies
 +
|- align="center"
 +
| D6tr || TRI || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| D7tr || TRI || 10<sup>-1</sup>|| overgrown
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-trbK_D8tr.jpeg D8tr] || TRI || 10<sup>-2</sup>|| <font color=green><B>6930</b></font>
 +
|- align="center"
 +
| D9tr || TRI || 10<sup>-3</sup>|| 720
 +
|- align="center"
 +
| D10tr || TRI || 10<sup>-4</sup>|| 81
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-trbK_T6tr.jpeg T6tr] || TRI + CAM || 10<sup>0</sup> || <font color=green><B>2</b></font>
 +
|- align="center"
 +
| T7tr || TRI + CAM || 10<sup>-1</sup>|| 0
 +
|- align="center"
 +
| T8tr || TRI + CAM || 10<sup>-2</sup>|| 0
 +
|- align="center"
 +
| T9tr || TRI + CAM || 10<sup>-3</sup>|| 0
 +
|- align="center"
 +
| T10tr || TRI + CAM || 10<sup>-4</sup>|| 0
 +
|- align="center"
 +
| R3tr || CAM || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| R4tr || CAM || 10<sup>-1</sup>|| 6333
 +
|}
 +
 +
also 3 positive control plates with no TrbK expression.
 +
 +
<br>
 +
{| border="1" align="center"
 +
|-
 +
| <b>Results</b>
 +
|- align="center"
 +
| Set # || Conjugation Efficinecy
 +
|- align="center"
 +
| 1 || 4.49E-6
 +
|- align="center"
 +
| 2 || 2.89E-6
 +
|- align="center"
 +
| control || 0.031
 +
|- align="center"
 +
|}
 +
 +
This successfully shows a reduction in the conjugation efficiency by four orders of magnitude when the entry exclusion protein is expressed.
 +
 +
 +
For the oriTR conjugation test:
 +
 +
{| border="1" align="center"
 +
|-
 +
| <b>Set 1</b>
 +
|- align="center"
 +
| Plate ID || Antibiotics || Dilution ||  # Colonies
 +
|- align="center"
 +
| D1ori || TRI + CAM || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| D2ori || TRI + CAM || 10<sup>-1</sup>|| overgrown
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_D3oriGFP.jpeg D3ori] || TRI + CAM || 10<sup>-2</sup>|| <font color=green><B>2939</b></font>
 +
|- align="center"
 +
| D4ori || TRI + CAM || 10<sup>-3</sup>|| 470
 +
|- align="center"
 +
| D5ori || TRI + CAM || 10<sup>-4</sup>|| 48
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_Ts1oriGFP.jpeg Ts1ori] || AMP + CAM || 10<sup>0</sup> || <font color=green><B>520</b></font>
 +
|- align="center"
 +
| Ts2ori || AMP + CAM || 10<sup>-1</sup>|| 1
 +
|- align="center"
 +
| Ts3ori || AMP + CAM || 10<sup>-2</sup>|| 0
 +
|- align="center"
 +
| Ts4ori || AMP + CAM || 10<sup>-3</sup>|| 0
 +
|- align="center"
 +
| Ts5ori || AMP + CAM || 10<sup>-4</sup>|| 0
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_tl1.jpeg TL1ori] || TRI + AMP || 10<sup>0</sup> || <font color=green><B>4092</b></font>
 +
|- align="center"
 +
| TL2ori || TRI + AMP || 10<sup>-1</sup>|| 802
 +
|- align="center"
 +
| TL3ori || TRI + AMP || 10<sup>-2</sup>|| 94
 +
|- align="center"
 +
| TL4ori || TRI + AMP || 10<sup>-3</sup>|| 7
 +
|- align="center"
 +
| TL5ori || TRI + AMP || 10<sup>-4</sup>|| 0
 +
|- align="center"
 +
| R1ori || AMP || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| R2ori || AMP || 10<sup>-1</sup>|| 5187
 +
|}
 +
 +
<br><br>
 +
{| border="1" align="center"
 +
|-
 +
| <b>Set 2</b>
 +
|- align="center"
 +
| Plate ID || Antibiotics || Dilution ||  # Colonies
 +
|- align="center"
 +
| D6ori || TRI + CAM || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| D7ori || TRI + CAM || 10<sup>-1</sup>|| overgrown
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_D8oriGFP.jpeg D8ori] || TRI + CAM || 10<sup>-2</sup>|| <font color=green><B>4950</b></font>
 +
|- align="center"
 +
| D9ori || TRI + CAM || 10<sup>-3</sup>|| 1066
 +
|- align="center"
 +
| D10ori || TRI + CAM || 10<sup>-4</sup>|| 114
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_Ts6oriGFP.jpeg Ts6ori] || AMP + CAM || 10<sup>0</sup> || <font color=green><B>1105</b></font>
 +
|- align="center"
 +
| Ts7ori || AMP + CAM || 10<sup>-1</sup>|| 12
 +
|- align="center"
 +
| Ts8ori || AMP + CAM || 10<sup>-2</sup>|| 0
 +
|- align="center"
 +
| Ts9ori || AMP + CAM || 10<sup>-3</sup>|| 0
 +
|- align="center"
 +
| Ts10ori || AMP + CAM || 10<sup>-4</sup>|| 0
 +
|- align="center"
 +
| TL6ori || TRI + AMP || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_tl7.jpeg TL7ori] || TRI + AMP || 10<sup>-1</sup>|| <font color=green><B>2251</b></font>
 +
|- align="center"
 +
| TL8ori || TRI + AMP || 10<sup>-2</sup>|| 352
 +
|- align="center"
 +
| TL9ori || TRI + AMP || 10<sup>-3</sup>|| 39
 +
|- align="center"
 +
| TL10ori || TRI + AMP || 10<sup>-4</sup>|| 7
 +
|- align="center"
 +
| R3ori || AMP || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| R4ori || AMP || 10<sup>-1</sup>|| 3961
 +
|}
 +
 +
<br><br>
 +
{| border="1" align="center"
 +
|-
 +
| <b>Set 3</b>
 +
|- align="center"
 +
| Plate ID || Antibiotics || Dilution ||  # Colonies
 +
|- align="center"
 +
| D11ori || TRI + CAM || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| D12ori || TRI + CAM || 10<sup>-1</sup>|| overgrown
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_D13oriGFP.jpeg D13ori] || TRI + CAM || 10<sup>-2</sup>|| <font color=green><B>4180</b></font>
 +
|- align="center"
 +
| D14ori || TRI + CAM || 10<sup>-3</sup>|| 737
 +
|- align="center"
 +
| D15ori || TRI + CAM || 10<sup>-4</sup>|| 46
 +
|- align="center"
 +
| Ts11ori || AMP + CAM || 10<sup>0</sup> || bad plate
 +
|- align="center"
 +
| Ts12ori || AMP + CAM || 10<sup>-1</sup>|| 3
 +
|- align="center"
 +
| Ts13ori || AMP + CAM || 10<sup>-2</sup>|| 0
 +
|- align="center"
 +
| Ts14ori || AMP + CAM || 10<sup>-3</sup>|| 0
 +
|- align="center"
 +
| Ts15ori || AMP + CAM || 10<sup>-4</sup>|| 0
 +
|- align="center"
 +
| TL11ori || TRI + AMP || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| [https://2009.igem.org/Image:Conjugation-aug18-oriTR_tl12.jpeg TL12ori] || TRI + AMP || 10<sup>-1</sup>|| <font color=green><B>1436</b></font>
 +
|- align="center"
 +
| TL13ori || TRI + AMP || 10<sup>-2</sup>|| 172
 +
|- align="center"
 +
| TL14ori || TRI + AMP || 10<sup>-3</sup>|| 18
 +
|- align="center"
 +
| TL15ori || TRI + AMP || 10<sup>-4</sup>|| 3
 +
|- align="center"
 +
| R5ori || AMP || 10<sup>0</sup> || overgrown
 +
|- align="center"
 +
| R6ori || AMP || 10<sup>-1</sup>|| 3679
 +
|}
 +
 +
<br>
 +
{| border="1" align="center"
 +
|-
 +
| <b>Results</b>
 +
|- align="center"
 +
| Set # || Conjugation Efficiency for Plasmid 1 || Conjugation Efficiency for wild R751
 +
|- align="center"
 +
| 1 || 0.00177 || 0.0141
 +
|- align="center"
 +
| 2 || 0.00224 || 0.0476
 +
|- align="center"
 +
| 3|| n/a || 0.0356
 +
|- align="center"
 +
|}
 +
 +
This shows that Conjugation Plasmid 1 is successfully transmitted but with a lower efficiency in the presence of R751.
 +
 +
 +
Attempted 2nd knockout
 +
 +
Made 4 0.5xKAN plates, 2 0.1xKAN plates, 2 1xTRI plates and 2 5mL tubes with 0.5xKAN.
 +
 +
Started centrifuging when R751 was at OD 0.507.
 +
 +
Used 7 &mu;L oriTR_PCR_KO vector for oriTR electroporation and 12 &mu;L trbK_KO_PCR vector for trbK electroporation. Both time constants 3.7. In incubator at 4:13 pm.
 +
 +
Modifications made to protocol:
 +
* Arabinose added the night before
 +
* used 4 mL from the 5 mL culture, higher density of cells in elctroporation
 +
* used 10% glycerol in the last spin down step
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Latest revision as of 10:09, 20 October 2009

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19 August 2009

Sriram

Today I did minipreps of all the biobricks I cultures yesterday. then again I continued with the assembly by digesting all the parts with the SpeI available in our lab. I am not very confident of it since the expiry date shows somewhere in 2008. So I thought let me continue the work properly after discussing with supervisors on friday because Daniel's backup plan biobricks are working very well.

Tim Weenink

After yesterdays horrible discovery that the I-SceI Homing Endonuclease we got from MIT was actually T4 DNA ligase we had to adjust our cloning strategy. The more primitive part without promoter was found to be the correct one, except that is was assembled with part !A instead of p(LacI). So we did that assembly again today. With p(LacI) from the delay team upstream, *T1 (downstream) and backbone pSB1C3 (also from the delay team). Both RbCl chemically comp and EC electrocomp cells were transformed.


Saeed

Saeed19082009wiki.jpg
lane Part Expected Plasmid Size Status
1 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]
2  !Fr1n upstream 1900 ok, but not needed
3  !Fr1n downstream 1900 ok
4 destination plasmid 3000 ok

!Fr1n downstream will be purified from the gel by gel extraction and used for the new stategy as Tim mentioned before.

Daniel

Digestion of biobricks:

Gel180809.jpg

Well Biobrick Expected Plasmid Size Status
1 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]
2 C0051 750 ✔
3 I13507 861 ✔
4 P0440 840 ✔
5 I13504 875 ✔
6 J04630 857 ✔
7 R0051 44 too small
8 B0015 129 ✔
9 J13002 74 ✔
10 pSB1C3 2072 ✔
11 pSB1AK3 3426 ✔
12 pSB1A3 2157 ✔
13 CB
14 CC
15 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]

Assemblies 1A, 2A, 3A, 4A, 5A and 6A

Transformation and culture in plates and tubes

Calin

Good results on the conjugation test. All plates were imaged on the safe imager with the 5 Megapixel camera. Colony counting was done with [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1770926 Clono-Counter].

For the trbK conjugation test:

Set 1
Plate ID Antibiotics Dilution # Colonies
D1tr TRI 100 overgrown
D2tr TRI 10-1 overgrown
D3tr TRI 10-2 4450
D4tr TRI 10-3 714
D5tr TRI 10-4 86
T1tr TRI + CAM 100 2
T2tr TRI + CAM 10-1 0
T3tr TRI + CAM 10-2 0
T4tr TRI + CAM 10-3 0
T5tr TRI + CAM 10-4 0
R1tr CAM 100 overgrown
R2tr CAM 10-1 8636



Set 2
Plate ID Antibiotics Dilution # Colonies
D6tr TRI 100 overgrown
D7tr TRI 10-1 overgrown
D8tr TRI 10-2 6930
D9tr TRI 10-3 720
D10tr TRI 10-4 81
T6tr TRI + CAM 100 2
T7tr TRI + CAM 10-1 0
T8tr TRI + CAM 10-2 0
T9tr TRI + CAM 10-3 0
T10tr TRI + CAM 10-4 0
R3tr CAM 100 overgrown
R4tr CAM 10-1 6333

also 3 positive control plates with no TrbK expression.


Results
Set # Conjugation Efficinecy
1 4.49E-6
2 2.89E-6
control 0.031

This successfully shows a reduction in the conjugation efficiency by four orders of magnitude when the entry exclusion protein is expressed.


For the oriTR conjugation test:

Set 1
Plate ID Antibiotics Dilution # Colonies
D1ori TRI + CAM 100 overgrown
D2ori TRI + CAM 10-1 overgrown
D3ori TRI + CAM 10-2 2939
D4ori TRI + CAM 10-3 470
D5ori TRI + CAM 10-4 48
Ts1ori AMP + CAM 100 520
Ts2ori AMP + CAM 10-1 1
Ts3ori AMP + CAM 10-2 0
Ts4ori AMP + CAM 10-3 0
Ts5ori AMP + CAM 10-4 0
TL1ori TRI + AMP 100 4092
TL2ori TRI + AMP 10-1 802
TL3ori TRI + AMP 10-2 94
TL4ori TRI + AMP 10-3 7
TL5ori TRI + AMP 10-4 0
R1ori AMP 100 overgrown
R2ori AMP 10-1 5187



Set 2
Plate ID Antibiotics Dilution # Colonies
D6ori TRI + CAM 100 overgrown
D7ori TRI + CAM 10-1 overgrown
D8ori TRI + CAM 10-2 4950
D9ori TRI + CAM 10-3 1066
D10ori TRI + CAM 10-4 114
Ts6ori AMP + CAM 100 1105
Ts7ori AMP + CAM 10-1 12
Ts8ori AMP + CAM 10-2 0
Ts9ori AMP + CAM 10-3 0
Ts10ori AMP + CAM 10-4 0
TL6ori TRI + AMP 100 overgrown
TL7ori TRI + AMP 10-1 2251
TL8ori TRI + AMP 10-2 352
TL9ori TRI + AMP 10-3 39
TL10ori TRI + AMP 10-4 7
R3ori AMP 100 overgrown
R4ori AMP 10-1 3961



Set 3
Plate ID Antibiotics Dilution # Colonies
D11ori TRI + CAM 100 overgrown
D12ori TRI + CAM 10-1 overgrown
D13ori TRI + CAM 10-2 4180
D14ori TRI + CAM 10-3 737
D15ori TRI + CAM 10-4 46
Ts11ori AMP + CAM 100 bad plate
Ts12ori AMP + CAM 10-1 3
Ts13ori AMP + CAM 10-2 0
Ts14ori AMP + CAM 10-3 0
Ts15ori AMP + CAM 10-4 0
TL11ori TRI + AMP 100 overgrown
TL12ori TRI + AMP 10-1 1436
TL13ori TRI + AMP 10-2 172
TL14ori TRI + AMP 10-3 18
TL15ori TRI + AMP 10-4 3
R5ori AMP 100 overgrown
R6ori AMP 10-1 3679


Results
Set # Conjugation Efficiency for Plasmid 1 Conjugation Efficiency for wild R751
1 0.00177 0.0141
2 0.00224 0.0476
3 n/a 0.0356

This shows that Conjugation Plasmid 1 is successfully transmitted but with a lower efficiency in the presence of R751.


Attempted 2nd knockout

Made 4 0.5xKAN plates, 2 0.1xKAN plates, 2 1xTRI plates and 2 5mL tubes with 0.5xKAN.

Started centrifuging when R751 was at OD 0.507.

Used 7 μL oriTR_PCR_KO vector for oriTR electroporation and 12 μL trbK_KO_PCR vector for trbK electroporation. Both time constants 3.7. In incubator at 4:13 pm.

Modifications made to protocol:

  • Arabinose added the night before
  • used 4 mL from the 5 mL culture, higher density of cells in elctroporation
  • used 10% glycerol in the last spin down step

Retrieved from "http://2009.igem.org/Team:TUDelft/19_August_2009"