Difference between revisions of "Part:BBa J23118"
Line 18: Line 18:
 
<partinfo>BBa_J23118 parameters</partinfo>
 
<partinfo>BBa_J23118 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
===Baltimore Biocrew 2019 Characterization===
 +
<strong>Goal</strong><br>
 +
 +
We, the Baltimore Biocrew, decided to characterize some of the Anderson promoters. These promoters are highly used by iGEM but the relative expression of these promoters have been routinely determined by measuring the fluorescence of a reporter protein. However, the function of a promoter is to start transcription of a gene so it may be more informative to measure the amount of RNA (instead of protein) produced by a reporter gene. Therefore, we decided to further characterize a selection of the Anderson promoters (J23100, J23101, J23103, J23105, J23118) by measuring RNA using Quantitative Polymerase Chain Reaction (qPCR).
 +
 +
<strong>Results</strong><br>
 +
 +
In our first trial of qPCR (8/03/19), we were able to measure the relative strengths for J23100, J23101, J23103, and J23105 which were 1.00, 0.00, 0.81, and 1.93, respectively. Since these strengths did not match the relative expression levels reported by iGEM2006_Berkeley, we repeated the qPCR (8/10/19) with the same cDNA. The strengths from this second trial were 1.00, 0.00, 0.37, and 0.20.  We repeated it again and the relative strengths that we got on 10/12/19 for J23100, J23101, J23103, and J23103 were 1, 0, 2.91, and .32. Next, we made new cDNA by growing new liquid cultures, extracting RNA again, and repeating reverse transcription. From the new cDNA, we repeated the qPCR procedure two more times. The relative strengths for that we got on 9/28/19 for J23100, J23101, J23103, J23105, and J23118 were 1, 24.63, .36, 1.76, and .25.  The relative strengths that we got on 10/12/19 for J23100, J23101, J23103, and J23105 were 1, 45.97, 3.20, and 1.26. In addition we measured promoter J23118 twice and got the strengths 1.13 and 1.32.
 +
 +
Here is the relative promoter strengths that we got from the qPCR.  Baltimore BioCrew in blue compared to the 2006 Berkeley iGEM in orange.
 +
[[File:Promoters_BaltimoreBiocrew2019.png|500px]]
 +
 +
To support our RNA measurements we also measured fluorescence of the liquid cultures we used to extract RNA. The cultures were grown overnight so we expected the bacteria to be at the stationary phase, but we measured OD to normalize any differences in growth.
 +
 +
 +
<html>
 +
 +
<style>
 +
table, th, td {
 +
  border: 1px solid black;
 +
  border-collapse: collapse;
 +
}
 +
th, td {
 +
  padding: 15px;
 +
}
 +
</style>
 +
</head>
 +
<body>
 +
 +
<table style="width:100%">
 +
<tr>
 +
      <th> Promoter </th>
 +
    <th>  OD  </th>
 +
    <th>fluorescence</th>
 +
    <th>fluorescence divided by OD</th>
 +
    <th>corrected relative expression</th>
 +
    <th>reported relative expression</th>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_J23100</td>
 +
    <td>0.876</td>
 +
    <td>250</td>
 +
    <td>285.38</td>
 +
    <td>1</td>
 +
    <td>1</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_J23101</td>
 +
    <td>0.674</td>
 +
    <td>255</td>
 +
    <td>378.33 </td>
 +
    <td>1.33</td>
 +
    <td>0.7</td>
 +
  </tr>
 +
    <tr>
 +
    <td>BBa_J23103</td>
 +
    <td>1.1</td>
 +
    <td>230</td>
 +
    <td>209.09 </td>
 +
    <td>0.73</td>
 +
    <td>0.01</td>
 +
  </tr>
 +
    <tr>
 +
    <td>BBa_J23105</td>
 +
    <td>1.08</td>
 +
    <td>215.74</td>
 +
    <td>209.09 </td>
 +
    <td>0.76</td>
 +
    <td>0.24</td>
 +
  </tr>
 +
      <tr>
 +
    <td>BBa_J23118</td>
 +
    <td>1.04</td>
 +
    <td>238</td>
 +
    <td>228.84 </td>
 +
    <td>0.80</td>
 +
    <td>0.56</td>
 +
  </tr>
 +
</table>

Revision as of 03:44, 20 October 2019

constitutive promoter family member

BerkiGEM2006-PromotersEppendorfs.jpg
BerkiGEM2006-Promoters.jpg

 
 Variant RFP (au)
 J23112           1
 J23103           17
 J23113           21
 J23109           106
 J23117           162
 J23114           256
 J23115           387
 J23116           396
 J23105           623
 J23110           844
 J23107           908
 J23106           1185
 J23108           1303
 J23118           1429
 J23111           1487
 J23101           1791
 J23104           1831
 J23102           2179
 J23100           2547
PBca1020-r0040.jpg

Constitutive promoter family
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. All parts except J23119 are present in plasmid J61002. Part J23119 is present in pSB1A2. This places the RFP downstream of the promoter. Reported activities of the promoters are given as the relative fluorescence of these plasmids in strain TG1 grown in LB media to saturation. See part BBa_J61002 for details on their use.

These promoter parts can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification. JCAraw

Saba: parts BBa_J23118 and BBa_J23110 are the same.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Baltimore Biocrew 2019 Characterization

Goal

We, the Baltimore Biocrew, decided to characterize some of the Anderson promoters. These promoters are highly used by iGEM but the relative expression of these promoters have been routinely determined by measuring the fluorescence of a reporter protein. However, the function of a promoter is to start transcription of a gene so it may be more informative to measure the amount of RNA (instead of protein) produced by a reporter gene. Therefore, we decided to further characterize a selection of the Anderson promoters (J23100, J23101, J23103, J23105, J23118) by measuring RNA using Quantitative Polymerase Chain Reaction (qPCR).

Results

In our first trial of qPCR (8/03/19), we were able to measure the relative strengths for J23100, J23101, J23103, and J23105 which were 1.00, 0.00, 0.81, and 1.93, respectively. Since these strengths did not match the relative expression levels reported by iGEM2006_Berkeley, we repeated the qPCR (8/10/19) with the same cDNA. The strengths from this second trial were 1.00, 0.00, 0.37, and 0.20. We repeated it again and the relative strengths that we got on 10/12/19 for J23100, J23101, J23103, and J23103 were 1, 0, 2.91, and .32. Next, we made new cDNA by growing new liquid cultures, extracting RNA again, and repeating reverse transcription. From the new cDNA, we repeated the qPCR procedure two more times. The relative strengths for that we got on 9/28/19 for J23100, J23101, J23103, J23105, and J23118 were 1, 24.63, .36, 1.76, and .25. The relative strengths that we got on 10/12/19 for J23100, J23101, J23103, and J23105 were 1, 45.97, 3.20, and 1.26. In addition we measured promoter J23118 twice and got the strengths 1.13 and 1.32.

Here is the relative promoter strengths that we got from the qPCR. Baltimore BioCrew in blue compared to the 2006 Berkeley iGEM in orange. Promoters BaltimoreBiocrew2019.png

To support our RNA measurements we also measured fluorescence of the liquid cultures we used to extract RNA. The cultures were grown overnight so we expected the bacteria to be at the stationary phase, but we measured OD to normalize any differences in growth.


Promoter OD fluorescence fluorescence divided by OD corrected relative expression reported relative expression
BBa_J23100 0.876 250 285.38 1 1
BBa_J23101 0.674 255 378.33 1.33 0.7
BBa_J23103 1.1 230 209.09 0.73 0.01
BBa_J23105 1.08 215.74 209.09 0.76 0.24
BBa_J23118 1.04 238 228.84 0.80 0.56