Contents 1 Gel Electrophoresis for the Amplified Fragments of rocF 1.1 Aim 1.2 Materials and Protocol 1.3 Result 1.4 Discussion 1.5 Conclusion 1.6 Solution for the problem 2 Amplification of the Pspac_oid promoter and RocF fragments by PCR 2.1 Aim 2.2 Materials and Protocol 2.2.1 PCR 2.3 Result 2.4 Discussion 2.5 Conclusion 2.6 Solution for the problem 3 Concrete Tensile Splitting Test 3.1 Aim 3.2 Materials 3.3 Procedure 3.4 Result 3.5 Discussion 3.6 Conclusion 4 Amplification of Pspac_oid promoter and lacI from a different stock of pMutin4 by PCR 4.1 Aim 4.2 Materials and Protocol 4.2.1 PCR 4.3 Discussion 4.4 Conclusion

Gel Electrophoresis for the Amplified Fragments of rocF

Aim

The aim of the experiment is to check for the PCR amplified RocF frangments that was performed on 4th August, 2010 by using gel electrophorsis.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

Figure 1: Gel electrophoresis of the pSB1C3, Pspac_oid promoter, rocF fragments and double terminator.

• Lane 1: 1kb DNA ladder
• Lane 2: BioBrick compatible vector pSB1C3
• Lane 3: Pspac_oid promoter
• Lane 4: 1st fragment of rocF CDS
• Lane 5: 2nd fragment of rocF CDS
• Lane 6: 3rd fragment of rocF CDS
• Lane 7: Double Terminator
• Lane 8: 1kb DNA ladder

	Biobrick compatible vector pSB1C3	Pspac_oid pormoter	1st fragment of rocF CDS	2nd fragment of rocF CDS)	3rd fragment of rocF CDS	Double Terminator
Size of the Fragment (in bp)	2072 approx.	106 approx.	246 approx.	597 approx.	125 approx.	116 approx.

Table 1: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

Correct sized bands were observed in lanes 2,4,5,6 and 7. However lane 3 did not contain any band.

Conclusion

PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was represented in lane 3. This could be due to the following prpeblems:

1. Primer sequences could be incorrect.
2. Melting temperature could be incorrect.
3. Plasmid pMutin4 could have degenerated due to long term storage.

Solution for the problem

1. Check the primer sequences so as to eliminate any problems associated with the primer sequence.
2. Perform PCR reactions for the Pspac_oid fragment with 3 different melting temperatures at 50°C, 51°C and 52°C.

Amplification of the Pspac_oid promoter and RocF fragments by PCR

Aim

The aim of this experiment is to amplify the Pspac_oid promoter fragment from the plasmid pMutin4 for the construction of the rocF BioBrick using 3 different melting temperatures in the Phusion PCR protocol, as well as to rerun the gel electrophoresis of the RocF fragments and the double terminator fragments obtained this morning.

Materials and Protocol

Please refer to PCR for the 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	Pspacoid Promoter	pMutin4	P1P1 forward	P2P1 reverse	51	106 approx.	15
2	Pspacoid Promoter	pMutin4	P1P1 forward	P2P1 reverse	50	106 approx.	15
3	Pspacoid Promoter	pMutin4	P1P1 forward	P2P1 reverse	52	106 approx.	15

Table 2: Table represents 3 different Phusion PCR reactions for the amplification of Pspac_oid promoter, 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. Therefore the extension time of each PCR reaction is different.
• To learn more about the rocF fragments, please refer to the Cloning strategy for rocF.

Result

Figure 2: Gel electrophoresis of the pSB1C3, Pspac_oid promoter, rocF fragments and double terminator.

• Lane 1: 100bp DNA ladder
• Lane 2: 1st fragment of rocF CDS
• Lane 3: 2nd fragment of rocF CDS
• Lane 4: 3rd fragment of rocF CDS
• Lane 5: Double Terminator
• Lane 6: Pspac_oid promoter (Tm 50°C i.e. Tube 2 of PCR reaction which is mentioned above)
• Lane 7: Pspac_oid promoter (Tm 51°C i.e. Tube 1 of PCR reaction which is mentioned above)
• Lane 8: Pspac_oid promoter (Tm 52°C i.e. Tube 3 of PCR reaction which is mentioned above)
• Lane 9: 100bp DNA ladder

	Pspac_oid pormoter	1st fragment of rocF CDS	2nd fragment of rocF CDS)	3rd fragment of rocF CDS	Double Terminator
Size of the Fragment (in bp)	106 approx.	246 approx.	597 approx.	125 approx.	116 approx.

Table 3: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

Correct bands size was observed in lanes 2,3,4 and 5 but not in lanes 6,7, and 8. The three different melting temperature used during the PCR for the Pspac_oid promoter is not successful.

Conclusion

The amplified frangments of RocF and the double terminator have been successful. However we are still not able to obtain the Pspac_oid promoter even when we have checked the primer sequence and using 3 different melting temperature. This could be due to the folloeing reason:

1. Plasmid pMutin4 could have degenerated due to long term storage.

Solution for the problem

1. Use a different stock of the plasmid pMutin4 and perform PCR reaction for the amplification of Pspac_oid promoter.

Concrete Tensile Splitting Test

Aim

To obtain samples of cracked concrete for BacillaFilla to fill up the cracks and also to determine the tensile strength of concrete before the cracks are filled up.

Materials

• Concrete cylinder
• Jubilee clips

Procedure

1. A concrete cylinder was made beforehand and left for more than 28 days to cure so that a straight line of crack will form down the diameter of the cylinder.
2. The cylinder is placed on two diametrically opposed loading generators. Two pieces of plywood are placed between the loading plates and the concrete cylinder to prevent failure in compression.
3. The generator is then started for loading until the cylinder forms a crack down the diameter. The maximum load is recorded and tensile strength of the concrete cylinder is calculated.

Concrete cylinder

Loading generator

Cracked concrete

Result

The maximum load that is recorded from the test is 171.3kN.

Discussion

From the formula f=(2P)/(πBD), where f=tensile strength, P=Maximum applied load, B=Depth of cylinder, D=Diameter of cylinder,

We calculated the maximum tensile strength of this concrete cylinder to be .

Conclusion

We now have the original tensile strength of the concrete cylinder, which is . We will test its tensile strength again after the concrete has been filled up by Bacilla Filla.

Amplification of Pspac_oid promoter and lacI from a different stock of pMutin4 by PCR

Aim

The aim of this experiment is to amplify the Pspac_oid promoter fragment and the lacI fragment from a different stock of plasmid pMutin4 using the Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 2 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	LacI	pMutin4	P1P1 forward	P2P1 reverse	54	1100 approx.	15
2	Pspacoid Promoter	pMutin4	P1P1 forward	P2P1 reverse	50	106 approx.	15

Table 4: Table represents 3 different Phusion PCR reactions for the amplification of Pspac_oid promoter from a different source of plasmid pMutin4, 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. Therefore the extension time of each PCR reaction is different.

Discussion

Gel electrophoresis was not done for the PCR amplification.

Conclusion

We will be using gel electrophoresis to check the outcome of both the PCR reactions on 6th August, 2010.