Contents 1 yneA 1.1 Gel extraction 1.1.1 Aims 1.1.2 Materials and Protocol 1.1.3 Results 1.1.4 Discussion 1.1.5 Conclusion 1.2 Ligation 1.2.1 Aims 1.2.2 Materials and Protocol 1.2.2.1 Ligation mix for pSB1C3 with yneA 1.2.3 Results and Conclusion 1.3 Digestion 1.3.1 Aim 1.3.2 Materials and Protocol 1.4 Setting up Overnight Cultures 1.4.1 Aims 1.4.2 Materials and Protocol 2 Subtilin Immunity 2.1 Aims 2.2 Materials and protocol 2.3 Discussion and Conclusion 3 rocF BioBrick 3.1 Gel extraction of linearised pSB1C3 3.1.1 Aims 3.1.2 Materials and protocol 3.1.3 Results 3.1.4 Discussion 3.1.5 Conclusion 3.2 PCR of pSB1C3, pspac oid and double terminator with new primers

yneA

Gel extraction

Aims

To purify the products of yneA, pGFPrrnB and PSB1AT3 by extracting the bands from the gel after running gel electrophoresis. Concentration of the DNA and vectors are then checked with nanodrop.

Materials and Protocol

Please refer to

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

Results

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

Discussion

The bands we extracted from the gel was good for pGFPrrnB, but not so strong for yneA and PSB1C3.

The results we got from the nanodrop were not as good as expected for yneA and PSB1C3.

Conclusion

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

Ligation

Aims

To ligate yneA into both vectors pGFPrrnB and pSB1C3.

Materials and Protocol

Please refer to ligation.

Ligation mix for pSB1C3 with yneA

The concentration of pSB1C3 and yneA that we got from the gel extraction earlier today was very low, so we used a different mixture set up for 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

Results and Conclusion

We will leave the ligation overnight because concentration of yneA, pGFPrrnB and pSB1C3 are not very good. Please refer to 20.08.10 for results and discussion.

Digestion

Aim

To repeat digestion from yesterday.

Materials and Protocol

Please refer to restriction digest.

Setting up Overnight Cultures

Aims

To prepare cultures for miniprep of yneA, pGFPrrnB and pSB1C3 tomorrow.

Materials and Protocol

Please refer to growing an overnight culture.

Subtilin Immunity

Aims

Today we received the four new primers that we ordered:

• Prom_For
• pSB1C3_for
• pSB1C3_rev
• Term_rev

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. Table # below shows the different fragments, primers and conditions used for each tube.

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. Thus the extension time of each and every PCR reaction is slightly different.
• The primers in bold are the new primers we received today. The primers not in bold were previously re-hydrated.

Discussion and Conclusion

Tommorrow we will carry out a test gel to check the sizes of the amplified fragments and if the sizes are correct then gel extraction will be performed

rocF BioBrick

Gel extraction of linearised pSB1C3

Aims

Following yesterday's Phusion PCR reactions, we plan to first perform gel electrophoresis of the amplified DNA sequences to check if they are of the correct sizes. If they appear to be successful we will then perform gel extraction, and finally nanodrop to record the level of DNA.

Materials and protocol

Please refer to the:

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

Results

Figure 1: Gel electrophoresis of the amplified PCR products

• Lane 1: 1 Kb ladder
• Lane 2: pSB1C3 (for Subtilin Immunity)
• Lane 3: pSB1C3 (for rocF)

• Lane 4: pVeg (for Subtilin Immunity)
• Lane 5: terminator (for Subtilin Immunity)
• Lane 6: pSpacoid (for rocF)
• Lane 7: terminator (for rocF)
• Lane 8: 100 bp ladder

Discussion

After looking at the results of our gel, there are no distinct band in lanes 2-3, therefore pSB1C3 vector did not work for both Subtilin Immunity or rocF. However, there are single bands in lanes 4-7 at similar positions to each other. They are about ... in size which is what is expected.

As lanes 2-3 showed no bands, we decided to make a range of Tms to perform another set of PCR reactions to check whether the Tm was the problem. Three Tms were used: 60°C, 65°C and 70°C and six tubes were set up; a duplicate was created for each Tm. After the PCR reactions were finished, there still wasn't any visible pSBIC3 vector band showing up. The problem could have occurred due to the working stocking solution for our primers. Our next step was to re-make the working stock solution for our primers. Also, we had decided to use the Tms 55°C, 60°C, 65°C, 70°C - duplicate for each, making eight PCR tubes in total.

As lanes 4-7 had bands at the right positions, gel extraction of these four parts was carried out. The concentration of DNA was then measured using the Nanodrop spectrophotometer.

Conclusion

The PCR machines were left to run and results of the eight PCR reactions at the 4 different temperatures shall be obtained on Monday. After a test gel has been run and if bands appear at the right sizes then gel extraction will be carried out.

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

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