Part:BBa_K4225013
Inhibitory Sequence (IS)
This is a new basic part that is made by HKUST Team 2022.
The inhibitory sequence (IS) is 77 amino acids from the C terminal of mRFP, placed downstream of the LVA tag sequence to shield it from being recognised and degraded from E. coli endogenous proteases ClpXP and ClpAp. Thereby creating a stable GFP that can be degraded when induced. A fluorescent assay using different concentrations of IPTG was performed to characterise the shielding ability of the inhibitory sequence
It is used to construct the composite parts:
Ptac-TEV -Pc-GFP-LVA-CS-IS (BBa_K4225019)
GFP-LVA-CS-IS (BBa_K4225017)
LVA-CS-IS (BBa_K4225014)
Experimental Design:
Constructs needed:
pTac - TEV - Pc - GFP - LVA - CS
pTac - TEV - Pc - GFP - LVA - CS - IS
Positive Control: pTac - TEV - Pc - GFP
Negative Control: pSB1C3
All constructs were successfully cloned, digestion checked and sequenced. For the negative control, we obtained an empty pSB1C3 vector by linearizing a DNA that has a pSB1C3 vector with XbaI and SpeI, and re-ligating the vector back. Digestion check is done to make sure the re-ligated pSB1C3 vector is correct.
Cells carrying the constructs were inoculated overnight and back diluted to a target 0.5 OD600 (log phase period). The cultures were grown at different concentrations of IPTG for 5 h The fluorescence tracked by a plate reader at excitation wavelength of 561.0nm and emission filter of 610.0nm, absorbance is also measured. Measurements are taken at 0 and 5 hours.
Results and discussion:
Duplicate measurements are done for the experiment and standard deviation of each point is shown as an error bar in the graph. In addition, fluorescence of each data is divided by the fluorescence of the blank, which is chloramphenicol LB, in order to obtain a normalised fluorescence.
As shown in figure 5.4, the last two concentration points, a decrease in fluorescence of the pTac - TEV - Pc - GFP - LVA - CS - IS construct can be observed in figure 5.3. Which suggests that the expression of TEV can remove the shielding effect of the inhibitory sequence, thereby inducing the degradation of GFP. Moreover, the construct pTac - TEV - Pc - GFP - LVA - CS remains at a low fluorescence level, indicating that the lack of an inhibitory sequence causes the construct to be continuously degraded. These 2 observations indicate that our constructs function well.
At low concentrations of IPTG, the fluorescence level of the test construct (pTac - TEV - Pc - GFP - LVA - CS - IS) is lower than that of the positive control (pTac - TEV - Pc - GFP).
This might be due to several reasons:
1) The inhibitory sequence did not fully shield GFP from degradation
- The shielding effect of the IS is dependent on the length of the amino acid sequence and its configuration. To increase shielding effect, more amino acids can be added to the IS or a completely different IS from other proteins can be used. A comparative assay with a similar setup can be conducted to test the functionality of different IS.
2) The addition of an inhibitory sequence interfered with the folding of GFP
- As the IS is sourced from 77 amino acids from the C terminal of mRFP and ligated directly to the back of the pTac - TEV - Pc - GFP - LVA - CS, the secondary and tertiary protein folding of the IS may cause steric interference/ collision to GFP, decreasing its fluorescent output. Therefore, we suggest that a longer spacer sequence can be inserted between the LVA tag and IS to aid in proper protein folding.
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]
| None |