Team:Newcastle/Chassis/LabStrategies

Lab Work Strategies
1. PCR up sleB and RBS using EcoRI and XbaI (Primer JJ1 – 5’) and SpeI (Primer JJ2 – 3’) as illustrated in Figure 1.



Labwork:

1.1 Perform PCR with Primer JJ1 and Primer JJ2 on wild type Bacillus subtilis, where the sleB region will be amplified. 1.2 Carry out DNA gel electrophoresis after the amplification of the DNA in Step 1.1, and we should see a fragment of approximately 918bp.

2. Cut pSB1AT3 or pSB1A2 (BioBrick compatible vector, already have it in stock) with EcoRI and SpeI, then purify backbone fragment using kit to get rid of mCherry.



Labwork:

2.1 Carry out restriction digest using the restriction enzymes EcoRI and SpeI, where the DNA segment mCherry is cut. 2.2 The DNA segment is then analysed via gel electrophoresis where the shorter fragment would be mCherry, and the longer fragment, the backbone fragment. 2.3 Use kit to process the backbone DNA fragment.



3. Ligate backbone fragment from Step 2, with PCR-ed sleB and RBS from Step 1, then cut with EcoRI and SpeI resulting in pJJ1.





4. PCR up cwlJ and RBS using EcoRI and XbaI as (Primer JJ3) and SpeI (Primer JJ4).



Labwork:

4.1 Perform PCR with Primer JJ3 and Primer JJ4 on wild type Bacillus subtilis, where the cwlJ region will be amplified. 4.2 Carry out DNA gel electrophoresis after the amplification of the DNA in Step 4.1, and we should see a fragment of approximately 426bp.

5. Purify and perform a midi prep for pJJ1 and cut with EcoRI and XbaI (restriction digest) to produce the fragment as seen in Figure 9.





Labwork:

5.1 Transform E.coli with pJJ1. 5.2 Conduct a mini prep. 5.3 Carry out gel electrophoresis. 5.4 Analyse results obtained from the gel electrophoresis. 5.5 If result is correct, carry out a midi prep to obtain lots of DNA. 5.6 Cut pJJ1 with restriction enzymes EcoRI and XbaI.

6. Ligate the product from Step 5 with PCR-ed cwlJ and RBS which were cut with EcoRI and SpeI, resulting in pJJ2 as seen in Figure 11.





7. Transform pJJ2, pick the correct colony and perform a mini prep to check

Labwork:

7.1 Transform E.coli with pJJ2. 7.2 Conduct a mini prep. 7.3 Carry out gel electrophoresis. 7.4 Analyse results obtained from the gel electrophoresis. 7.5 If result is correct, carry out a midi prep to obtain lots of DNA.

8. PCR the joined up sleB and cwlJ from pJJ2 using HindIII (Primer JJ5) and (Primer JJ6) BamHI primers.



8.1	Perform PCR with Primer JJ5 and Primer JJ6 on pJJ2, where the RBS + cwlJ and RBS + sleB region will be amplified. 8.2	Carry out DNA gel electrophoresis after the amplification of the DNA in Step 8.1.
 * Labwork:


 * Result:









9. Clone the joined up sleB and cwlJ from Step 8 into pMutin4 with HindIII and BamHI primers.
 * Cloning:





10. PCR pSpac:cwlJ:sleB from pMutin 4 with suitable primers for insertion into pGFP-rrnB. Suitable primers being EcoRI and XbaI (Primer PJJ7) and SpeI and PstI (Primer PJJ8).





11. Integrate


 * Testing and Characterisation:

We intend to use IPTG at difference concentrations to induce the promoter pSpac.