Our experimental plan is divided into three parts: the connection and expression of deoxycholic acid-responsive gene BreR with high expression promoter pGAP (TFBS) and mucin-binding protein gene MucBP with high expression promoter breAB in the engineered bacteria, as well as the verification of these two connections and the expression of the successfully sequenced plasmid in the engineered bacteria.

Our adhesion system consists of three main parts:

The input signal — High concentration of deoxycholic acid triggers the adhesion system

Signal pathway — BreR responds to deoxycholic acid and relieves transcriptional repression to facilitate the colonization of engineered bacteria in the colon

The output signal — Modified engineering bacteria were colonized in the colon

We plan to use two approaches to test the adhesion system.

In the first approach, we plan to use the Caco-2 cell line derived from human colon cancer cells as the substrate. By co-culturing the modified engineered bacteria with Caco-2 cells for 1-2 hours and measuring the fluorescence intensity, we will compare the results with those obtained when the unmodified engineered bacteria are cultured together. This comparison aims to validate the anticipated conclusion that the modified Escherichia coli DH5α demonstrates significantly stronger adhesion capability than its unmodified counterpart.

The Caco-2 cell line, derived from human colon cancer cells, can differentiate into a single-layered intestinal epithelial cell membrane under in vitro culture conditions, making it an ideal substrate for cellular colonization [7]. Fluorescence-labeled co-culture, as a well-established detection method, offers advantages such as clear results and rapid experimentation [8], thus being selected as the primary experimental protocol.

For the second approach, we developed a method using qualitatively treated filter paper with porcine gastric mucosal proteins as the substrate. After inoculating the bacterial suspension into sterile petri dishes with LB medium petri dishes at 37°C for 1-2 hours under aerobic conditions, the cultures were washed and spread across the solid medium surfaces in sterilized petri dishes. The modified engineered bacteria were incubated for 48-72 hours until colonies became clearly visible. Comparative analysis of colony counts between modified and unmodified strains confirmed that the engineered bacteria exhibited significantly enhanced adhesion capabilities compared to their original counterparts.

As a mucin expression gene, MucBP can colonize a specific location by binding to mucosal proteins. The growth of colonies also directly reflects adhesion ability of the bacterial strains. Therefore, this experiment was selected as the second experimental plan due to its simple operation and concise experimental steps. [4]

Through our efforts, we have achieved a series of results to validate our hypothesis. In the adhesion system section, we successfully developed engineered bacteria expressing BreR and MucBP genes separately, and conducted attempts to co-express both genes within a single Escherichia coli colony. Due to unfavorable experimental conditions in chemical transformation, we have not yet obtained a single colony containing both genes. However, we will continue optimizing the protocol in subsequent experiments to achieve success as soon as possible. Our experimental results and procedures are detailed below, providing insights into our research achievements.

The target gene was amplified via PCR and subjected to homologous recombination with the plasmid PMB1-A isolated from Bifidobacterium longum strain B2577, which had been digested by EcoR I and Xba I enzymes. The ligation product was then introduced into competent engineered bacteria for plate cultivation. After 12-16 hours, well-defined single colonies were selected for colony PCR analysis, followed by gel electrophoresis to verify the successful gene transfection. The experimental procedures including target gene amplification, enzyme digestion, plasmid recovery, and PCR gel electrophoresis are detailed below:

After completing the colony PCR and eliminating the false positive bacteria, we selected a single positive colony for the sequence detection, and successfully obtained the engineered bacteria containing pGAP+BreR and pGAP+MucBP respectively.

After receiving the two kinds of genetically engineered bacteria respectively, we carried out revival culture and transformation of the BreR gene engineered bacteria.

We chose to use Ca+ transformation method to chemically transform the engineered bacteria which were confirmed to contain the BreR gene with correct sequencing, and then introduce the plasmid containing MucBP gene into them to obtain the engineered bacteria containing both genes. However, some problems were encountered in this process.

During the first transformation, many microcolonies that are difficult to isolate grew on ourresistance plate, which we speculated were caused by the high concentration of calcium ions duringthe experiment". Therefore, we changed the 0.1 M/L CaCl2 used in the transformation to a mixture of0.08 M/L MgCl2 and 0.02 M/L CaCl2 and conducted the experiment again.

During the second chemical transformation, our bacterial colony number was too large to form a bacterial film covering the whole surface of the culture medium and single bacterial colonies could not be separated. After literature search and discussion, we believed that the high concentration of plasmid during transformation was the cause.

In response to this phenomenon, we controlled the amount of transferred plasmid during the second transformation and reduced it to 40% of the first transformation, but the result was still the formation of bacterial membrane. The control group also formed bacterial membrane, which proved that the main problem lies in the engineered bacteria used for preparing the receptive state.

Through further investigation, we concluded that the root cause likely lies in the resistance genes used by both MucBP and BreR genes being ampicillin resistance genes. During chemical transformation, engineered bacteria can be categorized into two groups: those without MucBP transfection and those with MucBP transfection. In reverse transcription culture, since the untransfected bacteria naturally possess resistance genes, their screening efficiency on ampicillin-resistant plates is significantly reduced. This results in both transfected and non-transfected engineered bacteria proliferating extensively, ultimately covering the entire surface of the resistance plate.

If we successfully construct an engineered bacterium containing both genes, we will test it using two methods we previously proposed: the Caco-2 cell assay and filter paper coated with mucosal protein assay. Here is our protocol: Adhesion System Protocol

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