Team:Caltech/Week 10

From 2011.igem.org

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CPCR of Gibson pNT002 and pNT003<br/>
CPCR of Gibson pNT002 and pNT003<br/>
Miniprep J23119 and I732019 to construct LacZ producer<br/>
Miniprep J23119 and I732019 to construct LacZ producer<br/>
-
GCMS of bisphenol A dissolved in methanol
+
GCMS of bisphenol A dissolved in methanol <br/>
 +
Gibson 16s sequencing DNA and vector together<br/>
===Results===
===Results===
CPCR gels showed 200 bp bands, indicating self-ligation of all colonies picked.<br/>
CPCR gels showed 200 bp bands, indicating self-ligation of all colonies picked.<br/>
Line 11: Line 12:
==August 16==
==August 16==
Electrospray bisphenol A dissolved in methanol<br/>
Electrospray bisphenol A dissolved in methanol<br/>
 +
Attempt to standard assemble constitutively expressed LacZ and transform into XL10 gold. However, there was no good way to select out the lacZ insert (I732019) from the backbone it was cut out of, as they are the same length.<br/>
 +
Nanodrop 16s solutions <br/>
===Results===
===Results===
Electrospray results show correct mass spectrometry peaks<br/>
Electrospray results show correct mass spectrometry peaks<br/>
 +
<table border="1">
 +
<tr>
 +
<th>Name</th>
 +
<th>concentration (ng/ul)</th>
 +
</tr>
 +
<tr>
 +
<td>9-3A</td>
 +
<td>35</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B</td>
 +
<td>47.9</td>
 +
</tr>
 +
<tr>
 +
<td>9-9A</td>
 +
<td>79.5</td>
 +
</tr>
 +
<tr>
 +
<td>9-9B</td>
 +
<td>50.4</td>
 +
</tr>
 +
<tr>
 +
<td>9-9C</td>
 +
<td>40.4</td>
 +
</tr>
 +
<tr>
 +
<td>10-3A</td>
 +
<td>31.1</td>
 +
</tr>
 +
</table>
 +
 +
==August 17==
 +
Borrowed HindIII from Nate to be able to gel extract lacZ from the now smaller backbone pieces<br/>
 +
Transformed psB1C3 into top10 cells<br/>
 +
===Results===
 +
The lacZ plated on LB-amp X-gal had about 520 colonies. The negative control had around 700 colonies. None of the colonies were blue, likely indicating that there was no functioning lacZ. Some were sent off for sequencing.<br/>
 +
Colonies for 16s sequencing! <br/>
 +
* 9-3A (1)
 +
* 9-3B (12)
 +
* 9-9A (1)
 +
* 9-9B (1)
 +
* 9-9C (0)
 +
* 10-3A (1)
 +
 +
==August 18==
 +
Restriction digest of R0010 and J23119 and their backbone for lacZ construct. Ligation of each promoter/backbone with lacZ purified yesterday. Transformed in XL-10 gold<br/>
 +
Prepare p450-degraded BPA for electrospray using organic extraction<br/>
 +
Start overnight cultures of 16s seequencing --> miniprep --> send off for sequencing <br/>
 +
Begin overnight cultures of WT-F87A<br/>
 +
===Results===
 +
4 colonies on pSB1C3 plate, all pink. <br/>
 +
==August 19==
 +
Visit Monk Hill Groundwater Treatment Plant<br/>
 +
Plate and sequence 16s vectors and WT-F87A<br/>
 +
Make glycerol stocks of WT-F87A<br/>
 +
Made new DH5a electrocompetent cells with Nate<br/>
 +
===Results===
 +
Electrospray of BPA in MeOH and its degraded products show a mass reduction of 22<br/>
 +
Nanodrop: <br>
 +
<table border="1">
 +
<tr>
 +
<th>Name</th>
 +
<th>concentration (ng/ul)</th>
 +
</tr>
 +
<tr>
 +
<td>9-3A 1</td>
 +
<td>32</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 1</td>
 +
<td>84.5</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 2</td>
 +
<td>46.7</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 3</td>
 +
<td>26.5</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 4</td>
 +
<td>57.5</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 5</td>
 +
<td>56.3</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 6</td>
 +
<td>70.1</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 7</td>
 +
<td>45</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 8</td>
 +
<td>59.9</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 9</td>
 +
<td>37.3</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 10</td>
 +
<td>29</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 11</td>
 +
<td>18.3</td>
 +
</tr>
 +
<tr>
 +
<td>9-3B 12</td>
 +
<td>52.6</td>
 +
</tr>
 +
<tr>
 +
<td>9-9A 1</td>
 +
<td>68.7</td>
 +
</tr>
 +
<tr>
 +
<td>10-3A 1</td>
 +
<td>17.6</td>
 +
</tr>
 +
<tr>
 +
<td>WT-F87A</td>
 +
<td>107.6</td>
 +
</tr>
 +
</table>
 +
Number of colonies on X-gal-ampicillin-LB plates
 +
<table border="1">
 +
<tr>
 +
<th>Name</th>
 +
<th>Number of colonies</th>
 +
</tr>
 +
<tr>
 +
<td>J23119+lacZ +</td>
 +
<td>~1100</td>
 +
</tr>
 +
<tr>
 +
<td>J23119+lacZ -</td>
 +
<td>25</td>
 +
</tr>
 +
<tr>
 +
<td>R0010+lacZ +</td>
 +
<td>~1800</td>
 +
</tr>
 +
<tr>
 +
<td>R0010+lacZ-</td>
 +
<td>57</td>
 +
</tr>
 +
</table>
 +
Most of the colonies on the positive plates were blue, indicating the presence of working lacZ.<br/>
 +
<gallery>
 +
File:R0010lacz.jpg|LacZ with inducible promoter in XL-10 gold cells
 +
File:J23119lacz.jpg|LacZ with constitutive promoter in XL-10 gold cells
 +
</gallery>
 +
==August 20==
 +
Visit West Basin Municipal Water Facility<br/>
 +
<gallery>File:DSC00440.jpg|Tour is a hard-hat because of the golf ball course next to West Basin
 +
File:DSC00446.jpg|Assistant general manager Shivaji Deshmukh explaining the water treatment process
 +
File:DSC00449.jpg|Walking up to the treatment tanks
 +
File:DSC00450.jpg|Ferric chloride and settling treatment
 +
File:DSC00458.jpg|Green tank: biological treatment; Red tank: ferric chloride
 +
File:DSC00465.jpg|Explaining microfiltration columns using bacteria plushie models
 +
File:DSC00467.jpg|Microfiltration units
 +
File:DSC00471.jpg|Reverse osmosis tanks
 +
File:DSC00474.jpg|Explaining reverse osmosis using more bacteria plushie models
 +
File:DSC00475.jpg|UV light treatment
 +
File:DSC00480.jpg|Taste test of treated water purity
 +
</gallery>
}}
}}

Latest revision as of 23:48, 25 August 2011


Caltech iGEM 2011



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August 15

CPCR of Gibson pNT002 and pNT003
Miniprep J23119 and I732019 to construct LacZ producer
GCMS of bisphenol A dissolved in methanol
Gibson 16s sequencing DNA and vector together

Results

CPCR gels showed 200 bp bands, indicating self-ligation of all colonies picked.
GCMS results show no correct mass spectrometry peaks.

August 16

Electrospray bisphenol A dissolved in methanol
Attempt to standard assemble constitutively expressed LacZ and transform into XL10 gold. However, there was no good way to select out the lacZ insert (I732019) from the backbone it was cut out of, as they are the same length.
Nanodrop 16s solutions

Results

Electrospray results show correct mass spectrometry peaks

Name concentration (ng/ul)
9-3A 35
9-3B 47.9
9-9A 79.5
9-9B 50.4
9-9C 40.4
10-3A 31.1

August 17

Borrowed HindIII from Nate to be able to gel extract lacZ from the now smaller backbone pieces
Transformed psB1C3 into top10 cells

Results

The lacZ plated on LB-amp X-gal had about 520 colonies. The negative control had around 700 colonies. None of the colonies were blue, likely indicating that there was no functioning lacZ. Some were sent off for sequencing.
Colonies for 16s sequencing!

  • 9-3A (1)
  • 9-3B (12)
  • 9-9A (1)
  • 9-9B (1)
  • 9-9C (0)
  • 10-3A (1)

August 18

Restriction digest of R0010 and J23119 and their backbone for lacZ construct. Ligation of each promoter/backbone with lacZ purified yesterday. Transformed in XL-10 gold
Prepare p450-degraded BPA for electrospray using organic extraction
Start overnight cultures of 16s seequencing --> miniprep --> send off for sequencing
Begin overnight cultures of WT-F87A

Results

4 colonies on pSB1C3 plate, all pink.

August 19

Visit Monk Hill Groundwater Treatment Plant
Plate and sequence 16s vectors and WT-F87A
Make glycerol stocks of WT-F87A
Made new DH5a electrocompetent cells with Nate

Results

Electrospray of BPA in MeOH and its degraded products show a mass reduction of 22
Nanodrop:

Name concentration (ng/ul)
9-3A 1 32
9-3B 1 84.5
9-3B 2 46.7
9-3B 3 26.5
9-3B 4 57.5
9-3B 5 56.3
9-3B 6 70.1
9-3B 7 45
9-3B 8 59.9
9-3B 9 37.3
9-3B 10 29
9-3B 11 18.3
9-3B 12 52.6
9-9A 1 68.7
10-3A 1 17.6
WT-F87A 107.6

Number of colonies on X-gal-ampicillin-LB plates

Name Number of colonies
J23119+lacZ + ~1100
J23119+lacZ - 25
R0010+lacZ + ~1800
R0010+lacZ- 57

Most of the colonies on the positive plates were blue, indicating the presence of working lacZ.

LacZ with inducible promoter in XL-10 gold cells

LacZ with constitutive promoter in XL-10 gold cells

August 20

Visit West Basin Municipal Water Facility

Tour is a hard-hat because of the golf ball course next to West Basin

Assistant general manager Shivaji Deshmukh explaining the water treatment process

Walking up to the treatment tanks

Ferric chloride and settling treatment

Green tank: biological treatment; Red tank: ferric chloride

Explaining microfiltration columns using bacteria plushie models

Microfiltration units

Reverse osmosis tanks

Explaining reverse osmosis using more bacteria plushie models

UV light treatment

Taste test of treated water purity


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