Team:Newcastle/Labwork/19 August 2009

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{{:Team:Newcastle/Header}}
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=Lab 19/08/09=
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__NOTOC__
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[[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]]
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=Formal Lab Session - 19th August 2009=
[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0484.JPG|400px|center]]
[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0484.JPG|400px|center]]
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==<u>Metal Sensing Team</u>==
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<br>
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=<font color="Orange"><u>Overview</u></font>=
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<font color="Orange">
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*[[#Metal Sensor Team|Metal Sensor Team]] '''- picked 3 colonies of potential ''BBa_J332066'' ''E. coli'' transformants and inoculated LB solution with them'''
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<br>
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*[[#Stochastic Switch Team|Stochastic Switch Team]] '''- only 5 out of 7 BioBricks had transformed in ''E. coli'' cells - these colonies were used to inoculate LB solution'''
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<br>
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*[[#Sporulation Tuning/Chassis Team|Sporulation Tuning/Chassis Team]] '''- counted the colonies produced by the treated sleB/cwlJ mutants'''
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</font>
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<br>
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==<u>Metal Sensor Team</u>==
===Introduction===
===Introduction===
So far in the lab, we have prepared the BioBrick ''BBa_J33206'' (''arsR'' promoter + coding sequence) from the repository and attempted transformation of this DNA in ''E.coli''. Once this procedure had been completed, the Metal Sensing team then streaked the bacteria on an LB + ampicillin plate and left it on 37°C incubation overnight.  
So far in the lab, we have prepared the BioBrick ''BBa_J33206'' (''arsR'' promoter + coding sequence) from the repository and attempted transformation of this DNA in ''E.coli''. Once this procedure had been completed, the Metal Sensing team then streaked the bacteria on an LB + ampicillin plate and left it on 37°C incubation overnight.  
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<br>
<br>
===Practical Outline===
===Practical Outline===
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[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0460.JPG|150px|thumb|Mathew about to pipette LB solution into flasks for overnights]]
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[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0460.JPG|150px|thumb|Mathew about to pipette 50ml LB solution into flasks for overnights]]
These are the steps that the Metal Sensing Team want to have completed by the end of the day:
These are the steps that the Metal Sensing Team want to have completed by the end of the day:
<br>
<br>
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===Observations and Procedure===
===Observations and Procedure===
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[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0481.JPG|200px|thumb|The three preparations of LB + ''Bacillus subtilis'' appeared cloudy indicating successful inoculations]]
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[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0481.JPG|200px|thumb|The three preparations of LB + ''E. coli'' appeared cloudy indicating successful inoculations]]
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[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0483.JPG|200px|thumb|Mathew preparing to inoculate 50ml LB with 50ml LB + ''E. coli'']]
When looking at the plate following an overnight incubation it could be seen that the transformation rate for the ''DH5α E.coli'' containing ''BBa_J33206'' DNA was very low – only five colonies were spotted on the plate.  
When looking at the plate following an overnight incubation it could be seen that the transformation rate for the ''DH5α E.coli'' containing ''BBa_J33206'' DNA was very low – only five colonies were spotted on the plate.  
Nevertheless three colonies were removed from the plate and, under aseptic conditions, used to inoculate 3 tubes containing 3ml LB each. These tubes were then placed in the shaking incubator at 37°C for the rest of the day. This step was done first thing in the morning. The bacteria which grow in these LB tubes will be used for a mini-prep of the ''BBa_J33206'' BioBrick tomorrow
Nevertheless three colonies were removed from the plate and, under aseptic conditions, used to inoculate 3 tubes containing 3ml LB each. These tubes were then placed in the shaking incubator at 37°C for the rest of the day. This step was done first thing in the morning. The bacteria which grow in these LB tubes will be used for a mini-prep of the ''BBa_J33206'' BioBrick tomorrow
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==<u>Stochastic Switch Team</u>==
==<u>Stochastic Switch Team</u>==
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[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0457.JPG|200px|thumb|Goksel and Jane have the task of analysing the plates for transformant cultures]]
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[[Image:Team Newcastle 2009 iGEM 19-08-09 IMG 0457.JPG|200px|thumb|Goksel and Jess have the task of analysing the plates for transformant cultures]]
===Summary===
===Summary===
Today we discovered that only 5 of 7 of our bricks had been transformed. Biobricks C0178 and C0062 had not worked. We decided to plate out 400ul of the remaining culture that we had left from yesterday onto a appropriate plates for both of these bricks in hope of getting some growth for miniprep. Today we also set up 3ml overnights for the remaining 5 bricks that worked (R0062; R0079; C0161; C0179; J44000). We did these in plain LB, later realising that we should maintain the selection for the Amp resistance plasmid by setting up the overnights in LB+antibiotic.
Today we discovered that only 5 of 7 of our bricks had been transformed. Biobricks C0178 and C0062 had not worked. We decided to plate out 400ul of the remaining culture that we had left from yesterday onto a appropriate plates for both of these bricks in hope of getting some growth for miniprep. Today we also set up 3ml overnights for the remaining 5 bricks that worked (R0062; R0079; C0161; C0179; J44000). We did these in plain LB, later realising that we should maintain the selection for the Amp resistance plasmid by setting up the overnights in LB+antibiotic.
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==<u>Sporulation Tuning/ Chassis Team</u>==
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==<u>Sporulation Tuning/Chassis Team</u>==
===Summary===
===Summary===
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[[Image:Newcastle 19 August Jane inspecting plate.jpg|thumb|Jane inspecting plate.]]
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[[Image:Newcastle 19 August sleB 1.jpg|thumb|sleB/cwlJ plate 1.]]
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Today, we came in to check out the plates for the ''sleB/cwlJ'' mutants which we treated [yesterday].
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[[Image:Newcastle 19 August sleB 101.jpg|thumb|sleB/cwlJ plate 10<sup>-1</sup>.]]
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[[Image:Newcastle 19 August sleB 102.jpg|thumb|sleB/cwlJ plate 10<sup>-1</sup>.]]
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In order to obtain accurate results, we counted the colonies, as can be seen in the following pictures.
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[[Image:Newcastle 19 August sleB count 1.jpg|thumb|counting colonies on the sleB/cwlJ plate 1.]]
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[[Image:Newcastle 19 August sleB count 101.jpg|thumb|counting colonies on the sleB/cwlJ plate 10<sup>-1</sup>.]]
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[[Image:Newcastle 19 August sleB count 1.jpg|thumb|250px|center|Counting colonies on the ''sleB/cwlJ'' mutants plate, concentration = 1.]]
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[[Image:Newcastle 19 August sleB count 102.jpg|thumb|counting colonies on the sleB/cwlJ plate 10<sup>-2</sup>.]]
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[[Image:Newcastle 19 August sleB count 101.jpg|thumb|250px|center|Counting colonies on the ''sleB/cwlJ'' mutants plate, concentration = 10<sup>-1</sup>.]]
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[[Image:Newcastle 19 August sleB noly.jpg|thumb|sleB/cwlJ no lysozyme plate.]]
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[[Image:Newcastle 19 August sleB count 102.jpg|thumb|250px|center|Counting colonies on the ''sleB/cwlJ'' mutants plate, concentration = 10<sup>-2</sup>.]]
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<br>
<br>
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<{{:Team:Newcastle/Project/Labwork/CalTemplate}}
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{{:Team:Newcastle/Footer}}
{{:Team:Newcastle/Footer}}
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{{:Team:Newcastle/Right}}

Latest revision as of 15:04, 20 October 2009


Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG

Formal Lab Session - 19th August 2009

Team Newcastle 2009 iGEM 19-08-09 IMG 0484.JPG


Overview

  • Metal Sensor Team - picked 3 colonies of potential BBa_J332066 E. coli transformants and inoculated LB solution with them


  • Stochastic Switch Team - only 5 out of 7 BioBricks had transformed in E. coli cells - these colonies were used to inoculate LB solution



Metal Sensor Team

Introduction

So far in the lab, we have prepared the BioBrick BBa_J33206 (arsR promoter + coding sequence) from the repository and attempted transformation of this DNA in E.coli. Once this procedure had been completed, the Metal Sensing team then streaked the bacteria on an LB + ampicillin plate and left it on 37°C incubation overnight.

Today’s procedure involves searching the plate for any colonies and if any colonies are present, picking three of them and inoculating 3ml LB with them. These grown up cultures will then be used for a mini-prep of the BBa_J33206 BioBrick. If no colonies are present, we must attempt transformation again. Because the plasmid involved in this BioBrick (pSB1A2) is a high copy plasmid, we can inoculate 3ml LB + ampicillin in the morning; by the afternoon the E.coli should have grown in the LB solution sufficiently to further inoculate 50ml LB solution in the afternoon (for midi-preps).

Practical Outline

Mathew about to pipette 50ml LB solution into flasks for overnights

These are the steps that the Metal Sensing Team want to have completed by the end of the day:

  1. Innoculate three tubes (each with 3ml of LB) with three of the E.coli cultures containing BBa_J33206 - morning
  2. Start preparing for a test PCR for the pGFP-rrnb plasmid in Bacillus subtilis.
  3. Should the E.coli cultures (containing BBa_J33206) have grown sufficiently in the LB tubes, further inoculate 3 flasks (containing 50ml of LB) with these bacteria – afternoon.


Observations and Procedure

The three preparations of LB + E. coli appeared cloudy indicating successful inoculations
Mathew preparing to inoculate 50ml LB with 50ml LB + E. coli

When looking at the plate following an overnight incubation it could be seen that the transformation rate for the DH5α E.coli containing BBa_J33206 DNA was very low – only five colonies were spotted on the plate. Nevertheless three colonies were removed from the plate and, under aseptic conditions, used to inoculate 3 tubes containing 3ml LB each. These tubes were then placed in the shaking incubator at 37°C for the rest of the day. This step was done first thing in the morning. The bacteria which grow in these LB tubes will be used for a mini-prep of the BBa_J33206 BioBrick tomorrow

In the afternoon, the team returned to the lab to observe the progress of the E.coli bacterial growth in the three LB tubes. The appearance of the three tubes was noted as cloudy which indicated the presence of sufficient numbers of E.coli bacteria. To save time in the labs, it was decided to inoculate 3 flasks (each containing 50ml LB) with these three samples – these 50ml flasks will then be used for midi-preps tomorrow should the mini-preps indicate that the E.coli have been successfully transformed.

To inoculate the 3 flasks containing 50ml LB each, the team took 0.5ml from each of the 3ml of cultures and pipette them into the separate flasks. These flasks were then placed into the orbital shaking incubator at 37°C overnight.

Stochastic Switch Team

Goksel and Jess have the task of analysing the plates for transformant cultures

Summary

Today we discovered that only 5 of 7 of our bricks had been transformed. Biobricks C0178 and C0062 had not worked. We decided to plate out 400ul of the remaining culture that we had left from yesterday onto a appropriate plates for both of these bricks in hope of getting some growth for miniprep. Today we also set up 3ml overnights for the remaining 5 bricks that worked (R0062; R0079; C0161; C0179; J44000). We did these in plain LB, later realising that we should maintain the selection for the Amp resistance plasmid by setting up the overnights in LB+antibiotic.

Sporulation Tuning/Chassis Team

Summary

Today, we came in to check out the plates for the sleB/cwlJ mutants which we treated [yesterday].

In order to obtain accurate results, we counted the colonies, as can be seen in the following pictures.

Counting colonies on the sleB/cwlJ mutants plate, concentration = 1.
Counting colonies on the sleB/cwlJ mutants plate, concentration = 10-1.
Counting colonies on the sleB/cwlJ mutants plate, concentration = 10-2.



<
July
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_July_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_July_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_July_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_July_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_July_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_July_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/7_July_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_July_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_July_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_July_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_July_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_July_2009&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_July_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/14_July_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_July_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_July_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_July_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_July_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_July_2009&action=edit 19]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_July_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_July_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_July_2009&action=edit 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_July_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_July_2009 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_July_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_July_2009&action=edit 26]
[http://2009.igem.org/Team:Newcastle/Labwork/27_July_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_July_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_July_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_July_2009 30] [http://2009.igem.org/Team:Newcastle/Labwork/31_July_2009 31]
August
MTWTFSS
          [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_August_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_August_2009&action=edit 2]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_August_2009&action=edit 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_August_2009 4] [http://2009.igem.org/Team:Newcastle/Labwork/5_August_2009 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_August_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_August_2009 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_August_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_August_2009&action=edit 9]
[http://2009.igem.org/Team:Newcastle/Labwork/10_August_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_August_2009 11] [http://2009.igem.org/Team:Newcastle/Labwork/12_August_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_August_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_August_2009 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_August_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_August_2009&action=edit 16]
[http://2009.igem.org/Team:Newcastle/Labwork/17_August_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_August_2009 18] [http://2009.igem.org/Team:Newcastle/Labwork/19_August_2009 19] [http://2009.igem.org/Team:Newcastle/Labwork/20_August_2009 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_August_2009 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_August_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_August_2009&action=edit 23]
[http://2009.igem.org/Team:Newcastle/Labwork/24_August_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_August_2009 25] [http://2009.igem.org/Team:Newcastle/Labwork/26_August_2009 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_August_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_August_2009 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_August_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_August_2009&action=edit 30]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_August_2009&action=edit 31]
September
MTWTFSS
  [http://2009.igem.org/Team:Newcastle/Labwork/1_September_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_September_2009 2] [http://2009.igem.org/Team:Newcastle/Labwork/3_September_2009 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_September_2009 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_September_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_September_2009&action=edit 6]
[http://2009.igem.org/Team:Newcastle/Labwork/7_September_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_September_2009 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_September_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_September_2009 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_September_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_September_2009&action=edit 13]
[http://2009.igem.org/Team:Newcastle/Labwork/14_September_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_September_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_September_2009 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_September_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_September_2009 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_September_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_September_2009&action=edit 20]
[http://2009.igem.org/Team:Newcastle/Labwork/21_September_2009 21] [http://2009.igem.org/Team:Newcastle/Labwork/22_September_2009 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_September_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_September_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_September_2009 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_September_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_September_2009&action=edit 27]
[http://2009.igem.org/Team:Newcastle/Labwork/28_September_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_September_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/Team:Newcastle/Labwork/1_October_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_October_2009 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_October_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_October_2009&action=edit 4]
[http://2009.igem.org/Team:Newcastle/Labwork/5_October_2009 5] [http://2009.igem.org/Team:Newcastle/Labwork/6_October_2009 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_October_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_October_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_October_2009 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_October_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_October_2009&action=edit 11]
[http://2009.igem.org/Team:Newcastle/Labwork/12_October_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_October_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_October_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_October_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_October_2009 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_October_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_October_2009&action=edit 18]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_October_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_October_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_October_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_October_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_October_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/24_October_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_October_2009&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_October_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_October_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/28_October_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_October_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_October_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_October_2009&action=edit 31]



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