Urease Test

Aims

The aim of this experiment was to determine if Bacillus subtilis 168 is able to produce urease and degrade urea.

Procedures

• Please refer to urease test

Inference

Christensen's Urea Agar was formulated to detect and differentiate urolytic and urea degrading microorganisms. The addition of gelatine peptone, dextrose and reduced content of buffer supports a luxuriant growth at an early stage. Urea is the substrate and can be degraded by certain organisms, which results in ammonia building. The ammonia makes the media alkaline and therefore the indicator phenol red will change from orange color to pink-red color.

• Negative control - No color change (Orange color)
• Test 1 (Duplicate) - Pink-red color
• Test 2 (Duplicate) - Pink-red color

For actual results, please see 02.07.10 lab notebook.

LacI BioBrick Construction

Aims

• To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (rfp).

Materials

• Competent E. coli (DH5α strain)
• pMutin4 plasmid
• pSB1AT3 plasmid

Protocol

• Please refer to: Transformation of E. coli DH5α with pMutin4 and pSB1AT3 separately.

Inference

• Ecoli DH5α is used as a bioreactor to replicate our plasmids to create a large stock ready for extraction from cells.

Competent E. coli Production

Aims

• To make a stock of competent E. coli (DH5α strain) ready for transformation.

Materials

• E. coli DH5α strain

Protocol

• Set up liquid culture consisting of E. coli DH5α.

Second transformation of 'Bacillius subtilis 168' with pGFP_rrnB containing YneA

Aim

The aim of the experiment is to insert the plasmid pGFP_rrnB containing yneA which have been ligated eariler, into the chromosome of Bacillus subtilis 168. B. subtilis containing the intergated vector will be resistance to both the antibiotic chloramphenicol and streptomycin, therefore those that have successfully intergated will be selected with agar plates that contain these antibiotic. The second step will be to identify those colones that have the plasmid intergated at the corerct position in the chromosome, which is the amylase gene locus. Thus those that have intergardted at the wrong position will not be able to break down starch, which can be tested with the iodine test.

Materials and Protocol

Please refer to: Transformation of Bacillus subtilis Note: Overnigth culture of 'B. subtilis 168' in MM competence medium was done the day before and the iodine test was performed the day after.

Result