Team:Caltech/Week 4

From 2011.igem.org

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Content=
Content=
__NOTOC__
__NOTOC__
-
==This Week==
+
==July 3==
-
<p>Try PCRing from source plate [http://partsregistry.org/Part:pSB4A5 pSB4A5] for Gibson<br/>
+
<p>Checked Gibson transformations<br/>
-
Gibson assemble pNT001 and pNT002 and negative controls following Joe's protocol.<br/>
+
Plated remaining transformed cells<br/>
-
Transform pNT001 and pNT002 into competent cells<br/>
+
Made new dilutions of original enrichment cultures (BPA, nonylphenol, 17-alpha-ethinyl-estradiol, DDT)</p>
-
Design experiments to test for degradation of BPA by pNT001 and pNT002 using the HPLC<br/>
+
 
-
Get the BioBricks we ordered from the parts registry ([http://partsregistry.org/Part:BBA_K364309 K364309], [http://partsregistry.org/Part:BBA_K364329 K364329], [http://partsregistry.org/Part:BBA_K364327 K364327], [http://partsregistry.org/Part:BBA_K364307 K364307])<br/>
+
===Results===
-
Transform these into cells, miniprep and sequence<br/>
+
There was no growth on the transformed Gibson plates. Since we did a small reaction, we used all of the reaction in transforming competent cells. The Gibson assembly likely failed, as one of the negative control plates had a single colony. However, we have had trouble with our competent cells in the past and will try plating what we have left.<br/><br/>
-
Continue with enrichment cultures<br/>
+
Gibson Transformations
-
Put LA River soil DNA into fosmids<br/>
+
<table border="1">
-
Work on Project Description and Safety Proposal<br/>
+
<tr>
-
Once we've sequenced ER ([http://partsregitry.org/Part:BBa_K123003 K123003]), start making test plasmids to see if it works.</p>
+
<th>Plate</th>
 +
<th>Number of Colonies</th>
 +
</tr>
 +
<tr>
 +
<td>pNT001 +</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 -</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT002 +</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT002 -</td>
 +
<td>1</td>
 +
</tr>
 +
</table>
 +
 
 +
==July 4==
 +
===Results===
 +
After plating the remaining 450 ul of our transformed cells:
 +
<table border="1">
 +
<tr>
 +
<th>Plate</th>
 +
<th>Number of Colonies</th>
 +
</tr>
 +
<tr>
 +
<td>pNT001 +</td>
 +
<td>10</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 -</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT002 +</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>pNT002 -</td>
 +
<td>0</td>
 +
</tr>
 +
</table>
 +
 
 +
==July 5==
 +
<p>Start overnight cultures of Gibson transformations<br/>
 +
Start gel to figure out length of purified river DNA (for fosmid kit)<br/>
 +
Confirm we have enough DNA for work with estrogen receptor<br/>
 +
Streak out new biobricks on chloramphenicol plates</p>
 +
 
 +
===Results===
 +
The gel of the river DNA was inconclusive, as no bands were visible (even the weight ladder).  The gel will be rerun.
 +
 
 +
==July 6==
 +
<p>Miniprep Gibson transformations and send off for sequencing<br/>
 +
Redo gel for fosmid kit<br/>
 +
Continue sequencing of estrogen receptor (using new forward primer and old reverse primer)<br/>
 +
Start overnight cultures of new biobricks<br/>
 +
Plate enrichment cultures on LB
 +
</p>
 +
 
 +
===Results===
 +
Again, no bands were visible on the gel.  We will run the samples on a smaller gel to check the size of the DNA and confirm that there is in fact DNA in the sample.
 +
 
 +
==July 7==
 +
<p>Miniprep new biobricks and send off for sequencing<br/>
 +
Meet with Pedro about HPLC and p450s<br/>
 +
Image enrichment culture plates<br/>
 +
Image second fosmid kit gel<br/>
 +
Run river DNA on small gel to check size<br/>
 +
Start new minimal media cultures from plates<br/>
 +
Design primers for estrogen receptor test construct<br/>
 +
 
 +
===Results===
 +
[[Image:River_gel_small.jpg|thumb|River samples run on a small gel.  1: ladder; 2:blank; 3: sample 9; 4: sample 10 ]]
 +
The smaller gel shows a smear at the top of the gel for one of the samples, indicating that this sample contains DNA that is probably of the correct size for work with the fosmid kit.<br/>
 +
 
 +
All of the plates of the enrichment cultures showed growth.
 +
<gallery caption="LB Plates of Enrichment Cultures">
 +
File:Ddtv.png|DDT with vitamins
 +
File:Ddtnov.png|DDT without vitamins
 +
File:Ethynylestradiolv.png|17a-ethynylestradiol with vitamins
 +
File:Ethynylestradiolnov.png|17a-ethynylestradiol without vitamins
 +
File:Nonylphenolv.png|Nonylphenol with vitamins
 +
File:Nonlyphenolnov.png|Nonylphenol without vitamins
 +
File:Bpavroom12.png|BPA with vitamins, room temperature, locations 1 and 2
 +
File:Bpavroom34.png|BPA with vitamins, room temperature, locations 3 and 4
 +
File:Bpanovroom12.png|BPA without vitamins, room temperature, locations 1 and 2
 +
File:Bpanovroom34.png|BPA without vitamins, room temperature, locations 3 and 4
 +
File:Bpav3012.png|BPA with vitamins, 30˚C, locations 1 and 2
 +
File:Bpav3034.png|BPA with vitamins, 30˚C, locations 3 and 4
 +
File:Bpanov3012.png|BPA without vitamins, 30˚C, locations 1 and 2
 +
File:Bpanov3034.png|BPA without vitamins, 30˚C, locations 3 and 4
 +
</gallery> </p>
 +
 
 +
==July 8==
 +
<p>Send off pNT001 and pNT002 for sequencing again <br/>
 +
Talk to Kenny about PFGE<br/>
 +
Work on design of test constructs for estrogen receptor</p>
 +
 
 +
==July 10==
 +
<p>Start overnight culture of pSB4A5 for use in construction of pNT003</p>
}}
}}

Latest revision as of 00:11, 12 July 2011


Caltech iGEM 2011



Home

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Data

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Human Impact

References

Support

July 3

Checked Gibson transformations
Plated remaining transformed cells
Made new dilutions of original enrichment cultures (BPA, nonylphenol, 17-alpha-ethinyl-estradiol, DDT)

Results

There was no growth on the transformed Gibson plates. Since we did a small reaction, we used all of the reaction in transforming competent cells. The Gibson assembly likely failed, as one of the negative control plates had a single colony. However, we have had trouble with our competent cells in the past and will try plating what we have left.

Gibson Transformations

Plate Number of Colonies
pNT001 + 0
pNT001 - 0
pNT002 + 0
pNT002 - 1

July 4

Results

After plating the remaining 450 ul of our transformed cells:

Plate Number of Colonies
pNT001 + 10
pNT001 - 0
pNT002 + 1
pNT002 - 0

July 5

Start overnight cultures of Gibson transformations
Start gel to figure out length of purified river DNA (for fosmid kit)
Confirm we have enough DNA for work with estrogen receptor
Streak out new biobricks on chloramphenicol plates

Results

The gel of the river DNA was inconclusive, as no bands were visible (even the weight ladder). The gel will be rerun.

July 6

Miniprep Gibson transformations and send off for sequencing
Redo gel for fosmid kit
Continue sequencing of estrogen receptor (using new forward primer and old reverse primer)
Start overnight cultures of new biobricks
Plate enrichment cultures on LB

Results

Again, no bands were visible on the gel. We will run the samples on a smaller gel to check the size of the DNA and confirm that there is in fact DNA in the sample.

July 7

Miniprep new biobricks and send off for sequencing
Meet with Pedro about HPLC and p450s
Image enrichment culture plates
Image second fosmid kit gel
Run river DNA on small gel to check size
Start new minimal media cultures from plates
Design primers for estrogen receptor test construct

Results

River samples run on a small gel. 1: ladder; 2:blank; 3: sample 9; 4: sample 10

The smaller gel shows a smear at the top of the gel for one of the samples, indicating that this sample contains DNA that is probably of the correct size for work with the fosmid kit.

All of the plates of the enrichment cultures showed growth.

LB Plates of Enrichment Cultures

DDT with vitamins

DDT without vitamins

17a-ethynylestradiol with vitamins

17a-ethynylestradiol without vitamins

Nonylphenol with vitamins

Nonylphenol without vitamins

BPA with vitamins, room temperature, locations 1 and 2

BPA with vitamins, room temperature, locations 3 and 4

BPA without vitamins, room temperature, locations 1 and 2

BPA without vitamins, room temperature, locations 3 and 4

BPA with vitamins, 30˚C, locations 1 and 2

BPA with vitamins, 30˚C, locations 3 and 4

BPA without vitamins, 30˚C, locations 1 and 2

BPA without vitamins, 30˚C, locations 3 and 4

July 8

Send off pNT001 and pNT002 for sequencing again
Talk to Kenny about PFGE
Work on design of test constructs for estrogen receptor

July 10

Start overnight culture of pSB4A5 for use in construction of pNT003


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