Part:BBa_K3189001
Phage Lambda-TetO
A modified phage lambda PL promoter with tet operator sites. This part functions as a promoter that allows for tetracycline-based transcriptional activation
GFP Expression using BBa_K3189001
The flasks in Figure 1 shows 50 mL E.coli cells transformed with gfp under the control of BBa_K3189001. On the plasmid containing BBa_K3189001 is the repressor, BBa_K3189004, needed for proper function of the promoter. Tetracycline was used to induce the system, resulting in GFP production and fluorescence is seen using a UV wand.
Figure 1: E. coli cells expressing GFP under the control of BBa_K3189001 induced with tetracycline.
An initial expression test of GFP under the control of BBa_K3189001 was preformed using 1 ng/mL and 50 ng/mL tetracycline. It can be seen in Figure 2 that more fluorescence was seen with the higher concentration of tetracycline. Based on these results it seems that BBa_K3189001 responds to tetracycline in a dose dependent manner.
Figure 2: E. coli strains DH5a and BL21(DE3) expressing GFP under the control of BBa_K3189001 using 1 ng/mL and 50 ng/mL tetracycline.
To determine what levels of tetracycline are needed to induce expression of GFP downstream of BBa_K3189001, a minimum inhibitory concentration (MIC) like test was preformed using decreasing level of tetracycline. The tetracycline levels used started at 10,000 ng/mL in the first column of the plate in Figure 3 and reduced by half in each subsequent well until reaching 0.01 ng/mL. This plate was imaged using an UV light table. Visually it can be seen that the brightest wells are at 312.5 ng/mL for E. coli BL21(DE3) with little to no fluorescence was observed E. coli DH5a.
Figure 3: GFP under the control of BBa_K3189001 expression at decreasing levels of tetracycline.
BBa_K3189001 in BBa_K3189015
The construct BBa_K3189015 containing the chromoprotein amilCP under the control of BBa_K3189001. When the system is induced with 100 ng/mL of tetracycline, a dark blue colour is produced (Figure 4 and Figure 5). This shows BBa_K3189001 is able to function with different reporter proteins other than just gfp.
Figure 4: amilCP under the control of BBa_K3189001. amilCP expression being induced using 100 ng/mL tetracycline (left) and uninduced (right).
Figure 5: Pellets of cells with amilCP under the control of BBa_K3189001 induced and uninduced with tetracycline. Pellet of cells induced with 100 ng/mL tetracycline (left) and pellet of cells uninduced (right).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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