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Contents 1 Gel extraction and ligation of yneA into pGFPrrnB and PSB1AT3 1.1 Aims 1.1.1 Materials and Protocol 1.1.2 Results 1.1.3 Discussion 1.1.4 Conclusion 1.1.5 Results and Conclusion 2 Rehydration of new subtilin immunity primers and PCR amplification 2.1 Aims 2.2 Materials and protocol 2.3 Discussion and Conclusion 3 Gel extraction of linearised pSB1C3 3.1 Aims 3.2 Materials and protocol 3.2.1 Discussion 3.2.2 Conclusion 3.3 PCR of pSB1C3, pspac oid and double terminator with new primers 3.3.1 Aim 3.3.2 Materials and Protocol 3.3.3 PCR 3.3.4 Discussion 3.3.5 Conclusion

Gel extraction and ligation of yneA into pGFPrrnB and PSB1AT3

Aims

The aim of the experiment is to further purify the yneA, pGFPrrnB and PSB1AT3 by doing gel extraction. The purified fragments are then ligated together.

Materials and Protocol

Please refer to gel electrophoresis, gel extraction, nanodrop spectrophotometer and ligation.

Ligation mix:

Reagents	1:3(μl)	1:5(μl)	Vector(μl)
Vector	3	2	1.5
Insert	3	6	7.5
10X BUFFER	1	1	1.1
T4 Ligase	1	1	1
H2O	2	0	0
Total Volume	10.0	10.0	10.0

Table 1: Ligation mix for ligation of yneA into pGFPrrnB and PSB1AT3

Results

	yneA 1	yneA 2	yneA 3	pSB1C3	pGFPrrnB
Concentration of DNA ng/µl	3.4	4.7	5.4	2.2	18.5

Table 2: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.

Discussion

The DNA concentration obtained for yneA ranged from 3.4 µl/ml to 5.4 µl/ml, 2.2 µl/ml for pSB1C3 and 18.5 µl/ml for pGFPrrnB . Therefore the concentration for yneA and pSB1C3 is low.

Conclusion

We will proceed with digestion, but another set of ligation will be set up to repeat the process again.

Results and Conclusion

Ligation will be done overnight because concentration of yneA, pGFPrrnB and pSB1C3 are not very good. Please refer to 20.08.10 for results and discussion. Overnight culture of yneA, pSB1C3 and pGFPrrnB were set up for miniprep extraction tomorrow, 20th August, 2010.

Rehydration of new subtilin immunity primers and PCR amplification

Aims

The aim of the experiment is to rehydrate the new primers, Prom_For, pSB1C3_for, pSB1C3_rev and Term_rev. The primers is then used to amplify the spaIFEG gene cluster, Promoter and RBS (pVeg-SpoVG) and Double terminator .

These primers were ordered as a solution to the problem we encountered before we were about to attempt carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. The primers will be used with previously rehydrated primers to try and obtain all four parts that we need to make the Subtilin Immunity BioBrick using the Gibson Protocol. So far the spaIFEG coding sequence (part 3) has already been successfully obtained. We still need to obtain the plasmid vector (part 1), promoter & RBS (part 2) and double terminator (part 4). So in order to do this the primers received today will be rehydrated and Phusion PCR will be carried out for each of the parts in order to amplify these parts.

Materials and protocol

Please refer to DNA rehydration page for the protocol for the rehydration of the four primers and to the Phusion PCR page for protocol followed for Phusion 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	68	2046 +	70
2	Promoter and RBS (pVeg-SpoVG)	BioBrick Bba_K143053	Prom_for	P2P2_rev	67	139 +	15
4	Double terminator	pSB1AK3 consisting BBa_B0014	P1T1_for	Term_rev	63	116 +	15

Table 1: This table shows the three different Phusion PCR reactions that were carried out today. If this is successful, these three parts (part 1, 2 and 4) along with (part 3) which is already obtained can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.

• The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
• The primers in bold are the new primers we received today. The primers not in bold were previously re-hydrated.

Discussion and Conclusion

Gel electrophoresis will be carried out tomorrow, 20th August, 2010 to check for the amplified spaIFEG gene cluster, Promoter and RBS (pVeg-SpoVG) and Double terminator.

Gel extraction of linearised pSB1C3

Aims

The aim of this experiment is to obtain linearised pSB1C3 plasmid by digesting it with restriction enzyme EcoR1 to linearize it and then to purify it by doing gel extraction.

Materials and protocol

Please refer to gel electrophoresis, gel extraction and NanoDrop spectrophotometer protocols.

Discussion

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 22 ng/µl concentration of plasmid.

PCR of pSB1C3, pspac oid and double terminator with new primers

Aim

The aim of this experiment is to amplify plasmid linearized pSB1C3, Pspac_oid promoter and double terminator 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	P1V1 forward	P2V1 reverse	65	2072 approx.	65
2	Pspacoid Promoter	pMutin4	Prom_forward	P2P1 reverse	67	106 approx.	15
3	Double Terminator	pSB1AK3 consisting BBa_B0014 biobrick	P1T1 forward	Term_reverse	63	116 approx.	15

Table 2: Table represents 3 different Phusion PCR reactions for the amplification of linearized plasmid pSB1C3, Pspac_oid promoter and double treminator 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.

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