Difference between revisions of "Team:Glasgow/Project/Overview/Terminator"
| Line 408: | Line 408: | ||
<h2>Methods</h2> | <h2>Methods</h2> | ||
| − | <p class="mainText">To characterise K1725081, the plasmid backbone <a href="http://parts.igem.org/Part:pSB1A10">pSB1A10</a> was used (figure 1). pSB1A10 contains I0500 (pBAD/<i>araC</i>), B0034 (strong ribosome binding site), E0040 (GFP), BioBrick prefix, BioBrick suffix, B0034, E1010 (RFP). With no terminator between the BioBrick prefix and suffix, cells transformed with the plasmid would fluoresce both green and red, however, when a terminator is inserted between the prefix and suffix, RFP expression is reduced, and the cells would fluoresce green. Plasmids for testing terminators were assembled by BioBrick Assembly: K1725081 in pSB1A10, <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a> (a double terminator consisting of B0010 and B0012; the most commonly used terminator in the registry, it is considered to be reliable) in pSB1A10, <a href="http://parts.igem.org/Part:BBa_B0010">B0010</a> (the terminator K1725081 is based on) in pSB1A10, and pSB1A10 with no terminator - the latter was made by ligating the XbaI and SpeI sites in the plasmid backbone prefix and suffix together (so the BioBrick cloning site became EcoRI-scar -PstI). | + | <p class="mainText">To characterise K1725081, the plasmid backbone <a href="http://parts.igem.org/Part:pSB1A10">pSB1A10</a> was used (figure 1). pSB1A10 contains I0500 (pBAD/<i>araC</i>), B0034 (strong ribosome binding site), E0040 (GFP), BioBrick prefix, BioBrick suffix, B0034, E1010 (RFP). With no terminator between the BioBrick prefix and suffix, cells transformed with the plasmid would fluoresce both green and red, however, when a terminator is inserted between the prefix and suffix, RFP expression is reduced, and the cells would fluoresce green. |
| + | <br> | ||
| + | <br> | ||
| + | Plasmids for testing terminators were assembled by BioBrick Assembly: K1725081 in pSB1A10, <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a> (a double terminator consisting of B0010 and B0012; the most commonly used terminator in the registry, it is considered to be reliable) in pSB1A10, <a href="http://parts.igem.org/Part:BBa_B0010">B0010</a> (the terminator K1725081 is based on) in pSB1A10, and pSB1A10 with no terminator - the latter was made by ligating the XbaI and SpeI sites in the plasmid backbone prefix and suffix together (so the BioBrick cloning site became EcoRI-scar -PstI). | ||
</br> | </br> | ||
</br> | </br> | ||
| − | <img style="text-align:center;height:70%;width:70%;" src="https://static.igem.org/mediawiki/2015/f/ff/Glasgow_2015_pSB1A10.png" | + | <center><img style="text-align:center;height:70%;width:70%;" src="https://static.igem.org/mediawiki/2015/f/ff/Glasgow_2015_pSB1A10.png"> |
| − | + | ||
| − | + | ||
</br> | </br> | ||
| + | <figcaption>Figure 1. The terminator testing plasmid used was pSB1A10.</figcaption> | ||
| + | </center></br> | ||
</br> | </br> | ||
Protocols for CaCl<sub>2</sub> competent cells, transformation, miniprep, restriction digest, gel electrophoresis, ethidium bromide staining, Azure A staining, gel extraction, oligo annealing, and ligation available on our <a href="https://2015.igem.org/Team:Glasgow/Project/Overview/Protocols">Protocols</a> page. Fluorescence measurements taken as documented on our <a href="https://2015.igem.org/Team:Glasgow/Interlab">Interlab Study</a> page. | Protocols for CaCl<sub>2</sub> competent cells, transformation, miniprep, restriction digest, gel electrophoresis, ethidium bromide staining, Azure A staining, gel extraction, oligo annealing, and ligation available on our <a href="https://2015.igem.org/Team:Glasgow/Project/Overview/Protocols">Protocols</a> page. Fluorescence measurements taken as documented on our <a href="https://2015.igem.org/Team:Glasgow/Interlab">Interlab Study</a> page. | ||
| Line 425: | Line 428: | ||
</br> | </br> | ||
</br> | </br> | ||
| + | <center> | ||
<img style="text-align:center;height:70%;width:70%;" src="https://static.igem.org/mediawiki/2015/f/f6/Glasgow_2015_Terminator_Scan_and_Graph.png"> | <img style="text-align:center;height:70%;width:70%;" src="https://static.igem.org/mediawiki/2015/f/f6/Glasgow_2015_Terminator_Scan_and_Graph.png"> | ||
</br> | </br> | ||
| − | < | + | <figcaption>Figure 2. Characterising K1725081. All terminators in pSB1A10 backbone, in DH5α cells. Three replicates of the sample were diluted and tested under the same conditions for each sample. Mean and standard deviation of replicates were calculated to give value and error bars.</figcaption> |
| − | </br></p> | + | </center></br></p> |
Latest revision as of 18:28, 14 November 2015
Terminator
Home > Project > Terminator
Summary
Aim: To characterise the T1 terminator from E. coli rrnB, K1725081, for submission to the registry. Results Overview: K1725081 was successful in terminating transcription to the same level as B0010 and B0015. Basic Parts submitted: • BBa_K1725081
Introduction
In prokaryotes, such as E. coli, terminators occur at the end of a coding region or operon, and stop transcription. K1725081 was used in our Bistable Switch.
Methods
To characterise K1725081, the plasmid backbone pSB1A10 was used (figure 1). pSB1A10 contains I0500 (pBAD/araC), B0034 (strong ribosome binding site), E0040 (GFP), BioBrick prefix, BioBrick suffix, B0034, E1010 (RFP). With no terminator between the BioBrick prefix and suffix, cells transformed with the plasmid would fluoresce both green and red, however, when a terminator is inserted between the prefix and suffix, RFP expression is reduced, and the cells would fluoresce green.
Plasmids for testing terminators were assembled by BioBrick Assembly: K1725081 in pSB1A10, B0015 (a double terminator consisting of B0010 and B0012; the most commonly used terminator in the registry, it is considered to be reliable) in pSB1A10, B0010 (the terminator K1725081 is based on) in pSB1A10, and pSB1A10 with no terminator - the latter was made by ligating the XbaI and SpeI sites in the plasmid backbone prefix and suffix together (so the BioBrick cloning site became EcoRI-scar -PstI).
Results
The results of the fluorescence scan are shown in Figure 2. K1725081, B0015, and B0010 all show reduced RFP expression, indicating that they are successfully stopping transcription after GFP - although neither of these terminator stop 100% of transcription, so there is still some RFP expression for all three.
Read More!
