Part:BBa_K4786000
Promoter_piliA_V.n
The promoter of Vibrio natriegens PilA gene. Its activity can reflect the expression level of the PilA gene. For instance it can be combined with a reporter gene like GFP so as to form a reporter system.
Brief introduction
We wish to express the highly conductive pilus of Geobacter metallireducens in Vibrio natriegens. The energy required to produce such protein nanowires from Vibrio natriegens is over two orders of magnitude less than that required to produce silicon or carbon nanowires of the same mass, and the production process does not produce any toxic chemicals.
Since Vibrio natriegens has its own pilus,which can become impurities in the production process of Geobacter metallireducens pilus, we hope to introduce dCas9 to inhibit the expression of Vibrio natriegens pilA by CRISPRi technology so as to achieve the goal of harvesting a solution containing only Geobacter metallireducens pilus.
We used this Part to construct a plasmid that utilizes the pilA Promoter of Vibrio natriegens to initiate downstream sfGFP expression, hoping to use this system to determine the effect of CRISPRi inhibition by measuring either the expression level of sfGFP or the fluorescence intensity, which would then allow us to adjust the expression of the CRISPRi system in order to find the right parameters to achieve the inhibition of Vibrio natriegens pilA expression.
Characterization
SDS-PAGE
1 mL of overnight V.GF4 culture and 1 mL of overnight ATCC14048 culture were centrifuged and then resuspended with 35 uL of ddH2O. 1.5 mL of overnight V.GF4 culture and 1.5 mL of overnight ATCC14048 culture were used for soluable protein extraction. The result of SDS-PAGE was as follows.
There was a significantly darker band on the lane of V.GF4 soluable proteins(marked with).The molecular weight of the modified sfGFP is about 31kD, between 25kD and 35kD. The experiment would be repeated two more times to ensure the result.
Fluorescence absorption
After modification of the coding sequence, we acquired two other versions of report plasmid pGFPcut and pGFPmod2.Overnight culture of wild type Vibrio natriegens and strain V.mod, V.cut were spread out on appropriate agar plates.The plates were cultured at 37 ℃ before harvest.The cells were collected with LB medium and the OD600 of three suspensions were adjusted to the same.Green fluorescence of the suspension was measured with microplate reader and the result was as follows.It could be seen that there was stronger green fluorescence in strain V.cut and V.mod. This further showed that Promoter_pilA_V.n could be used to express other proteins in Vibrio natriegens.
We produced a total of four plasmids, pdCas9-sg1-4 and then co-transformed with pGFPmod2 respectively. After IPTG induced dCas9 for 5 hours, the inhibition of GFP expression by dCas9 was shown by fluorescence values. Green fluorescence of the suspension was measured with microplate reader and the result was as follows.
From the measured results, it can be seen that all CRISPRi plasmids have some inhibition effect except for pdCas9-sg1, for which the result is not significant, and pdCas9-sg4 has the best inhibition effect. However, due to time constraints, we only used the initial pdCas9sg-1 plasmid for the construction of the bacterial hair expression strain.
Generally we identified three effective CRISPRi target sites in this promoter.
- 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 |