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Contents 1 yneA 1.1 Gel Electrophoresis 1.1.1 Aim 1.1.2 Materials and Protocol 1.1.3 Results 1.1.4 Discussion 1.1.5 Conclusion 1.2 Transformation of B. subtilis 1.2.1 Aim 1.2.2 Materials and Protocol 1.2.3 Results and Conclusion 1.3 Transformation 1.3.1 Aim 1.3.2 Materials and Protocol 1.3.3 Results and Conclusion 1.4 PCR 1.4.1 Aim 1.4.2 Materials and Protocol 1.4.3 Conclusion 1.5 PCR Purification 1.5.1 Aim 1.5.2 Materials and Protocol 1.6 Digestion 1.6.1 Aim 1.6.2 Materials and Protocol 1.7 Gel extraction 1.7.1 Aim 1.7.2 Materials and Protocol 1.7.3 Results 1.7.4 Conclusion 2 rocF and Subtilin immunity 2.1 Gel extraction 2.1.1 Aims 2.1.2 Materials and protocol 2.1.3 Results 2.1.4 Discussion 2.1.5 Conclusion 2.2 PCR 2.2.1 Aim 2.2.2 Materials and Protocol 2.2.3 PCR 2.2.4 Discussion 2.2.5 Conclusion

yneA

Gel Electrophoresis

Aim

To check if the digestion from yesterday worked.

Materials and Protocol

Please refer to gel electrophoresis for protocol.

Results

The result from gel electrophoresis:

Figure 1 shows the double digest of 12 tubes of pGFPrrnB and yneA.

• Lane 1: Vector only
• Lane 2: Tube 1
• Lane 3: Tube 2
• Lane 4: Tube 3
• Lane 5: Tube 4
• Lane 6: Tube 5
• Lane 7: Tube 6
• Lane 8: Tube 7
• Lane 9: Tube 8
• Lane 10: Tube 9
• Lane 11: Tube 10
• Lane 12: Tube 11
• Lane 13: Tube 12

Discussion

The bands we got from the gel shows that digestion in tubes 1, 2, 4, 10, 11 and 12 worked.

Conclusion

We use the digested products from the six tubes that worked for ligation.

Transformation of B. subtilis

Aim

To transform yneA into competent B. subtilis.

Materials and Protocol

Please refer to transformation of B. subtilis.

Results and Conclusion

Please refer to results in tomorrow's lab book.

Transformation

Aim

To transform pGFPrrnB and yneA into E. coli.

Materials and Protocol

Please refer to transformation of E. coli.

Results and Conclusion

Please refer to tomorrow's lab book.

PCR

Aim

To amplify the DNA pSB1C3 using RocF primer.

Materials and Protocol

Please refer to PCR.

Conclusion

Continue with PCR purification.

PCR Purification

Aim

To remove unwanted primers, taq polymerase, buffer and salts to obtain pure DNA.

Materials and Protocol

Please refer to PCR purification.

Digestion

Aim

To digest the PCR products of pSB1C3 and yneA from PCR purification.

Materials and Protocol

Please refer to restriction digest.

Gel extraction

Aim

To purify the DNA of yneA and pSB1C3 by extracting the bands from the gel after running gel electrophoresis. Concentration of DNA is then checked with NanoDrop.

Materials and Protocol

Please refer to:

• gel electrophoresis,
• gel extraction and
• nanodrop spectrophotometer.

Results

The bands we got from gel electrophoresis is very faint.

Conclusion

We realized that we used the wrong rocF primer, so we repeat the whole procedure from PCR again.

rocF and Subtilin immunity

Gel extraction

Aims

We digested the plasmid pSB1C3 with the restriction enzyme HindIII to linearize it and then we would be running the sequence on the gel to check if the plasmid has become linear or not and then we would be extracting it.

Materials and protocol

Please refer to the:

• gel electrophoresis,
• gel extraction and
• NanoDrop spectrophotometer protocols.

Results

• Lane 1: 1 Kb ladder
• Lane 2: Linearized plasmid pSB1C3
• Lane 3: 1 Kb ladder

There is no gel photograph because we want to keep the exposure of DNA to the UV light to an absolute minimum.

Discussion

During gel extraction procedure, we found a bright band of approx During gel extraction procedure, we found a bright band of approximately 3100 bp size in lane 2 under UV light and we cut the gel and extracted the band.

Conclusion

We got linearized plasmid pSB1C3 and we performed gel extraction successfully and the nanodrop protocol showed that we got 12.7 ng/µl concentration of plasmid.

PCR

Aim

The aim of this experiment is to amplify plasmid linearized pSB1C3 at 3 different melting temperatures for the construction of rocF BioBrick with the help of 3 different Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:

PCR

Tube	Part to be amplified	DNA fragment consisting the part	Forward primer	Reverse Primer	Melting Temperature (Tm in °C)	Size of the fragment (in bp)	Extension time* (in seconds)
1	Plasmid Vector	pSB1C3	pSB1C3_for	pSB1C3_rev	64	2072 approx.	65
2	Plasmid Vector	pSB1C3	pSB1C3_for	pSB1C3_rev	65	2072 approx.	65
3	Plasmid Vector	pSB1C3	pSB1C3_for	pSB1C3_rev	66	2072 approx.	65

Table 1: Table represents 3 different Phusion PCR reactions for the amplification of linearized plasmid pSB1C3 at three different melting temperatures so that it can be ligated together with other fragments for the construction of rocF with the help of Gibson Cloning method.

• The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
• For more about the rocF fragments, please refer to the Cloning strategy for rocF.

Discussion

All the 3 Phusion PCR reactions were done however, gel electrophoresis will be done tomorrow, to check whether the fragments have actually amplified or not.

Conclusion

Tomorrow we will be running gel electrophoresis to check the outcome of the 3 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.