Difference between revisions of "Part:BBa K3257045"

Revision as of 03:58, 6 October 2019

Improved LacI

LacI is one of the genes in Lac operon encoding the inhibitor protein binding to LacO sites (cis-acting element). In response to IPTG, the inhibitor protein detaches from LacO and enables the transcription of downstream genes. We mutated some specific sites in the LacI gene to improve its sensibility to IPTG.^[1] Using EGFP as a reporter, its fluorescence intensity appears a lower leakage and the same level of expression before and after the induction of IPTG. Also, we induce the improved Lac operon by arabinose to verify its orthogonal response to IPTG.

This part is improved in the following three main aspects.

Lower Response to Arabinose, Better Orthogonality

Cross talk between the response to IPTG and arabinose has been a defect of the wild type Lac operon. When 4mM arabinose added, a few lac inhibitors detach from lac operator which means that it is induced in a relatively low but unignorable level. According to the measurement of our experiment, our improved LacI can respond to IPTG with better orthogonality. The figure below (Figure 1) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. It shows that when 4mM arabinose is added, oLacI(wild-type LacI) is induced at a significantly high level while iLacI(improved LacI) is induced at a lower level.

Same Level of Expression as the Wild-type Lac operon when Induced by IPTG

Next step we prove that our improved Lac operon can be normally induced by IPTG, which counts a great deal for the usage of this operon. The figure below (Figure 2) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. It shows that when 1mM IPTG is added, EGFP controlled by both operons can be disinhibited and expressed at a relatively same level.

Lower Uninduced Leakage

The uninduced leakage level is also an important parameter of an operon. Improved LacI lowers the leakage level compared to the wild-type one. The figure below (Figure 3) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. When no IPTG or arabinose is added, the fluorescence of EGFP controlled by improved Lac operon is under the detection range while the fluorescence of EGFP controlled by wild-type Lac operon remains a detectable signal indicating a considerably undesired leakage.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Reference

[1] AJ Meyer et al. Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2019 Feb;15(2):196-204. doi: 10.1038/s41589-018-0168-3. Epub 2018 Nov 26.

@@ Line 8: / Line 8: @@
 '''Lower Response to Arabinose, Better Orthogonality'''
-'''Same Level of Expression when Induced by IPTG'''
+Cross talk between the response to IPTG and arabinose has been a defect of the wild type Lac operon. When 4mM arabinose added, a few lac inhibitors detach from lac operator which means that it is induced in a relatively low but unignorable level. According to the measurement of our experiment, our improved LacI can respond to IPTG with better orthogonality. The figure below (Figure 1) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. It shows that when 4mM arabinose is added, oLacI(wild-type LacI) is induced at a significantly high level while iLacI(improved LacI) is induced at a lower level.
-'''Lower Uninduced Leakage'''
+'''Same Level of Expression as the Wild-type Lac operon when Induced by IPTG'''
+Next step we prove that our improved Lac operon can be normally induced by IPTG, which counts a great deal for the usage of this operon. The figure below (Figure 2) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. It shows that when 1mM IPTG is added, EGFP controlled by both operons can be disinhibited and expressed at a relatively same level.
+'''Lower Uninduced Leakage'''
+The uninduced leakage level is also an important parameter of an operon. Improved LacI lowers the leakage level compared to the wild-type one. The figure below (Figure 3) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. When no IPTG or arabinose is added, the fluorescence of EGFP controlled by improved Lac operon is under the detection range while the fluorescence of EGFP controlled by wild-type Lac operon remains a detectable signal indicating a considerably undesired leakage.