Team:Newcastle/Chassis/LabStrategies

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Meetings Calendar

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.

Figure 1: RBS + sleB

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.

Figure 2: Cut mCherry out of the plasmid backbone (pSB1AT3 or pSB1A2)

Figure 3: Remaining backbone fragment after cutting out 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.

Figure 4: Plasmid part BBa_J04450 (pSB1AT3)

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.

Figure 5: Ligating backbone fragment and PCR-ed RBS and sleB

Figure 6: pJJ1 - Ligated backbone fragment and PCR-ed RBS and sleB

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

Figure 7: RBS and cwlJ

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.

Figure 8: Cutting pJJ1 with restriction enzymes ECoRI and XbaI

Figure 9: pJJ1 fragment awaiting for the RBS and cwlJ fragment

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.

Figure 10: Ligating PCR-ed RBS and cwlJ with remaining pJJ1 fragment

Figure 11: pJJ2 – Ligated PCR-ed RBS and cwlJ with pJJ1 fragment

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.

Figure 12: RBS and cwlJ, and RBS and sleB fragment

Labwork

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, and we should see a fragment of approximately ___bp.

Result

Figure 13: RBS and sleB sequence

Figure 14: sleB cut sites

Figure 15: RBS and cwlJ sequence

Figure 16: cwlJ cut sites

Cloning

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

Figure 17: Ligating the RBS and cwlJ, and RBS and sleB segment into pMutin4

Figure 18: pMutin4

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).

Figure 19: pSpac:cwlJ:sleB fragment

Figure 20: pGFP-rrnB

11. Integrate

Testing and Characterisation

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

20 – 21 June 2009 - Europe workshop (London)
23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
23 October Practice Presentation (Newcastle)
23 October T-shirts are ready
27 October Practice Presentation (Sunderland)
27 October Poster is ready
30 October – 2 November 2009 - Jamboree (Boston)

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