csgD and OmpR234 Dual Expression

This composite part consists of a strong promoterBBa_J23118,a strong RBS BBa_B0034,transcriptional regulators of two curli operons - csgD BBa_K805015 and OmpR234 BBa_K342003 and a double terminator BBa_B0015. The strong promoter and strong RBS are used for the overexpression of the desired proteins. By overexpressing both the proteins, curli fiber production in Escherichia coli K-12 can be increased.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 877
1000
COMPATIBLE WITH RFC[1000]

Characterization

In this dual expression construct, the upregulation of OmpR234 activates the csgD gene. The csgD gene undergoes transcription followed by translation and finally csgD protein is produced. csgD is the positive transcriptional regulator and it controls the expression of curli proteins csgA, csgB and csgC.

Our new composite part BBa_K4509469 has been created to increase the percentage growth in the bacteria. The part BBa_K2229300 submitted by TAS Taipei iGEM 2017 consists of BBa_J23100 promoter. Though the promoter strength of J23100 is high compared to the other promoters, the burden value is J23100 is also high.The burden value of J23100 IS 20.0% ± 9.9%. Since the bacteria is producing csgD protein as well as OmpR234 protein, the dual expression of the genes along with the promoter burden should not affect the lifespan of the bacteria. Hence, we decided to reduce the burden value and improve the quality of the bacteria by introducing BBa_J23118 promoter instead of BBa_J23100 into the construct. The burden value of J23118 is only -0.1% ± 3.5%.

Assays

The efficiency of the biofilm will indicate the nature of the parts added. To check this, we performed the following three assay: Congo red assay, Crystal violet assay and Safranin assay.

CONGO RED ASSAY

Take 5mL of the bacterial culture in a 15mL tubes.Pellet down the cells at 13000 rpm for 5 min.Remove the supernatant.Add 3mL of the Congo Red solution.Let incubate for 10 min. Pellet down the cells at 13000 rpm for 5 min.Remove the supernatant.The absorbance was measured at 500 nm.Compare the results with the positive and negative control.

CRYSTAL VIOLET ASSAY

The culture was diluted in the ratio of 1:100 with 195 μl of fresh LB broth and 5ul of culture in the well.The plates were then transferred to thermostatic static incubators maintained at 25℃. After 24 hrs, the 96 well plate was taken from the incubator and the planktonic cells were removed.The wells were then washed using sterile PBS.Each well was then added with 200 μl of 0.1% crystal violet and was kept for 15 min for staining.The crystal violet was then removed and washed with sterile PBS and kept the plate for air drying for 5 min.The stained biofilm was then solubilised by adding 200 μl of 30% acetic acid.The plate was then kept in a microplate reader and OD value was recorded at 570 nm.

SAFRANIN ASSAY

Add 200μl of diluted 2nd overnight culture into each well (96 well plates) and incubate at proper conditions overnight.Discard the overnight bacteria and wash with Ethanol.Add 200μl 0.1% safranin per well to stain the biofilm.Wait for a couple of minutes, then discard unabsorbed safranin and wash once with ethanol.Add 200 μl of 30% acetic acid into each well and wait for 5 minutes.Measure absorbance of samples at OD of 530 nm.

From the figures and graphs, we can observe that E.coli strain being a poor biofilm forming strain did not produce biofilm well, E.coli with BBa_K2229300 produced strong biofilm and E.coli with BBa_K4509469 produced biofilm but the congo red color was less intense compared to the color produced for E.coli with BBa_K2229300.

Growth Curve