"
Team:UNIPV-Pavia/Project/PromotoriAuto
From 2010.igem.org
(→TOGLIMI!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!) |
(→TOGLIMI!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!) |
||
| Line 452: | Line 452: | ||
|'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''Scell ratio LB'''||'''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''||'''Scell ratio M9''' | |'''Self-inducible device''' || '''Description''' || '''O.D.start LB''' || '''K_HSL LB'''|| '''Doubling time LB'''|| '''Scell ratio LB'''||'''O.D.start M9''' || '''K_HSL M9'''|| '''Doubling time M9'''||'''Scell ratio M9''' | ||
|- | |- | ||
| - | |<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo> | + | |<partinfo>BBa_K300017</partinfo> (wiki name: I7) in <partinfo>pSB4C5</partinfo> plasmid |
|[[Image:pv_BBa_K300017.png|400px]] | |[[Image:pv_BBa_K300017.png|400px]] | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
| - | | | + | | <font color='red' size='+2'>X</font> |
| - | | | + | | 0.24 <br> ± <br> 0.0004 ** |
| - | | | + | | 8.35 10^-17 <br> ± <br> 3.62 10^-18 ** |
| - | | | + | | 62.41 <br> ± <br> 1.56 ** |
| - | | | + | | 0.08 <br> ± <br> 0.008 ** |
|- | |- | ||
| - | |<del style='color:#C2DFFF'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo> | + | |<del style='color:#C2DFFF'><partinfo>BBa_K300014</partinfo> (wiki name: I8) in <partinfo>pSB4C5</partinfo> plasmid</del> |
|[[Image:pv_BBa_K300014.png|400px]] | |[[Image:pv_BBa_K300014.png|400px]] | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
| Line 474: | Line 474: | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
|- | |- | ||
| - | |<del style='color:#C2DFFF'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo> | + | |<del style='color:#C2DFFF'><partinfo>BBa_K300015</partinfo> (wiki name: I9) in <partinfo>pSB4C5</partinfo> plasmid</del> |
|[[Image:pv_BBa_K300015.png|400px]] | |[[Image:pv_BBa_K300015.png|400px]] | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
| Line 485: | Line 485: | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
|- | |- | ||
| - | |<del style='color:#C2DFFF'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo> | + | |<del style='color:#C2DFFF'><partinfo>BBa_K300016</partinfo> (wiki name: I10) in <partinfo>pSB4C5</partinfo> plasmid</del> |
|[[Image:pv_BBa_K300016.png|400px]] | |[[Image:pv_BBa_K300016.png|400px]] | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
| Line 496: | Line 496: | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
|- | |- | ||
| - | |<del style='color:#C2DFFF'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo> | + | |<del style='color:#C2DFFF'><partinfo>BBa_K300012</partinfo> (wiki name: I12) in <partinfo>pSB4C5</partinfo> plasmid</del> |
|[[Image:pv_BBa_K300012.png|400px]] | |[[Image:pv_BBa_K300012.png|400px]] | ||
| <font color='red' size='+2'>X</font> | | <font color='red' size='+2'>X</font> | ||
Revision as of 14:53, 26 October 2010
|
Self-inducible promotersRegulation of signal protein productionExperimental implementation: <partinfo>BBa_K300009</partinfo> part was assembled downstream of different constitutive promoters, thus obtaining a signal molecule generator. The choice of constitutive promoters was performed between the ones belonging to the [http://partsregistry.org/Part:BBa_J23101 Anderson’s promoters collection] ; we chose promoters according to their activities reported in the Registry of Standard Biological Parts, in order to have a thick mesh:
Before constructing the signal generators, <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> under the regulation of one of these constitutive promoters, we evaluated the promoter activities in Relative Promoter Units (R.P.U.) according to Data analysis for RPU evaluation, using the reporter protein RFP (Red Fluorescent Protein) in different experimental conditions (plasmids’ copy number and growth medium), many of them not yet explored and documented:
It was not possible to evaluate promoters activities in low copy number plasmids and LB because the RFP activity was too weak and not distinguishable from the background. RFP fluorescence and Optical Density at 600nm (O.D.600) were measured in 96-well microplates, as reported in Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2 and data were analyzed as reported in Data Analysis RPU; Results: results are shown here.
Discussion: we observed that the ranking previously documented in the Registry is not valid in all the conditions, even if a general agreement can be observed. As an example, <partinfo>BBa_J23110</partinfo> in high copy plasmid is stronger than <partinfo>BBa_J23118</partinfo>, in contrast with the ranking reported in the Registry. After the evaluation of promoter activity, signal generators were constructed in high copy and low copy plasmids: <partinfo>BBa_K300009</partinfo> and <partinfo>BBa_K300010</partinfo> were assembled downstream of the above mentioned promoters, thus obtaining the following parts:
Some of the promoters could not be cloned upstream of these devices because they produced LuxI protein amounts that give a high metabolic burden for E. coli, so it was not possible to study all the combinations as transformans could not be obtained in some cases. For each part, a measurement system was built, exploiting the production of the reporter gene GFP (Green Fluoresent Protein) to evaluate the "switch on" condition of every self-inducible promoter. Many different combinations were explored, in order to provide a library of promoters able to initiate transcription at the desired culture density. Quantification of the HSL producedExperimental implementation The new parts were, thus, characterized, measuring the HSL concentration released in the medium after a 6 hour growth of the cultures. All the details are available in this section. <partinfo>BBa_T9002</partinfo> contained in <partinfo>pSB1A3</partinfo> in E. coli TOP10 was used as a HSL->GFP biosensor. In every experiment, a HSL-GFP calibration curve with known concentration of HSL was produced. Results The amount of 3OC6-HSL produced after a 6 hours growth by E. coli DH5alpha bearing the parts contained in high copy plasmid <partinfo>pSB1A2</partinfo> is reported in Fig.8 and in the table:
The amount of 3OC6-HSL produced after 6 hours growth by the parts contained in low copy plasmid <partinfo>pSB4C5</partinfo> is reported in Fig.9 and in the table:
Discussion These experiments provided extremely useful informations about the capability of the signal generators to produce the 3OC6-HSL signal molecule. Data are quantitative, but incomplete because for weak promoters or medium-strength promoters contained in a low copy number plasmid the amount of 3OC6-HSL was not detectable using this system. However, this simple experiment shows that there is a strong correlation between the strength of promoter and the amount of signal molecule produced. These results confirm that the production of the autoinducer can be engineered in E. coli and different expression systems reach different amounts of 3OC6-HSL in the growth media as a function of the promoter strength. Thus, these results demonstrate that self-inducible circuits can be rationally designed from a set of well characterized standard parts. Modulation of plasmid copy numberSignal generator and sensor device were assembled in an unique part (such as <partinfo>BBa_K300017</partinfo>, <partinfo>BBa_K300014</partinfo>, <partinfo>BBa_K300015</partinfo>, <partinfo>BBa_K300016</partinfo> and <partinfo>BBa_K300012</partinfo>) beared on high copy number plasmid <partinfo>pSB1A2</partinfo> or low copy number plasmid <partinfo>pSb4C5</partinfo>. A third alternative was the assembly of signal generator on a low copy number plasmid (<partinfo>pSB4C5</partinfo>) and the receiver device on high number plasmid (<partinfo>pSB1A2</partinfo>). The circuits we obtained and tested are summarized here:
ResultsThe following measurement systems were realized assembling GFP downstream of each self-inducible device. The parts characterized are reported in this table:
Cultures of E. coli TOP10 bearing the plasmids containing the self-inducible devices expressing G.F.P. were grown according to this protocol and all data collected were analyzed as explained in this section
Doubling times were estimated as explained here Thus, these BioBrick parts can be used to express recombinant proteins without adding an inducer to trigger the transcription of their genes; in large-scale production of such proteins this strategy could be also cost saving. For every self-inducible device, several parameters were evaluated, as reported in this section. Results are summarized in the following tables:
TOGLIMI!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Tab. 1 - Sender and Receiver on high copy plasmid <partinfo>pSB1A2</partinfo>
Tab. 2 - Sender and Receiver on low copy plasmid <partinfo>pSB4C5</partinfo>
Tab. 3 - Sender on low copy plasmid <partinfo>pSB4C5</partinfo> and Receiver on high copy plasmid <partinfo>pSB1A3</partinfo>
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
