Difference between revisions of "Part:BBa K3292001"

Latest revision as of 02:17, 22 October 2019

T7-LacO Promoter + strong RBS + sialidase + sfGFP + 6x his-tag + double terminator

This device uses Bba_K2406020, Bba_J34801 and Bba_0010 to regulate the expression of a super folding GFP (Bba_I746916). The main objective of creating this device is to facilitate the secretion and characterization of any protein intracellularly produced after adding its nucleotide sequence next to the sfGFP.

Characterization

The characterization of this part included a Bradford assay to quantify the amount of protein present in the medium for each of the proposed constructs. The conditions for the assay are listed below.

The other characterization assay was a fluorescence measurement, where bacteria were growth at 30ºC for 3 hrs. Then 100µl of the grown bacteria were induced with IPTG at 0, 40, 200 and 400 mMm microtiter florescent measures were taken every half an hour for a period of 3 hours. Absorption and emission values were 395/509 nm.

Figure 1. Bradfford calibration curve

Figure 2. Bradford assay for the proteins

Figure 3. sfGFP grpah, fluorescence vs time induced at different concentrations.

Figure 4. sial+sfGFP grpah, fluorescence vs time induced at different concentrations.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 721
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1986
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 646
Illegal BsaI.rc site found at 510
Illegal BsaI.rc site found at 1647
Illegal SapI.rc site found at 2055

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 The other characterization assay was a fluorescence measurement, where bacteria were growth at 30ºC for 3 hrs. Then 100µl of the grown bacteria were induced with IPTG at 0, 40, 200 and 400 mMm microtiter florescent measures were taken every half an hour for a period of 3 hours. Absorption and emission values were 395/509 nm.
-<div class = "left">
-https://2019.igem.org/wiki/images/3/38/T--Tec-Monterrey--part-bradford-sial-.png
-</div>
-Figure 1. Bradford calibration curve
 <div class = "left">
 https://2019.igem.org/wiki/images/thumb/3/38/T--Tec-Monterrey--part-bradford-sial-.png/800px-T--Tec-Monterrey--part-bradford-sial-.png
 </div>
-Figure 2. Bradfford calibration curve
+Figure 1. Bradfford calibration curve
 <div class = "left">
 https://2019.igem.org/wiki/images/thumb/7/75/T--Tec-Monterrey--part-bradford-sial2-.png/800px-T--Tec-Monterrey--part-bradford-sial2-.png
 </div>
-Figure 3. Bradford assay for the proteins
+Figure 2. Bradford assay for the proteins
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 https://2019.igem.org/wiki/images/thumb/3/30/T--Tec-Monterrey--demostrate-grafica.png/800px-T--Tec-Monterrey--demostrate-grafica.png
 </div>
-Figure 4. sfGFP grpah, fluorescence vs time induced at different concentrations.
+Figure 3. sfGFP grpah, fluorescence vs time induced at different concentrations.
 <div class = "left">
 https://2019.igem.org/wiki/images/thumb/3/32/T--Tec-Monterrey--part-sfGFP-sial-.png/800px-T--Tec-Monterrey--part-sfGFP-sial-.png
 </div>
-Figure 5. sial+sfGFP grpah, fluorescence vs time induced at different concentrations.
+Figure 4. sial+sfGFP grpah, fluorescence vs time induced at different concentrations.