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Team:Newcastle/31 August 2010
From 2010.igem.org
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Contents |
Amplification of the circular plasmid pSB1C3 for the Subtilin immunity BioBrick
Aim
The aim of this experiment is to amplify the plasmid pSB1C3 for the construction of the subtilin immunity BioBrick with the help of two Phusion PCR reactions using old and new primers, and to perform gel electrophoresis to check whether amplification was successful.
Materials and Protocol
Please refer to PCR for Phusion PCR protocol and gel electrophoresis. The details for the PCR reactions are given 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 | 58 | 2072 approx. | 65 |
| 2 | Plasmid Vector | pSB1C3 | pSB1C3_for | pSB1C3_rev | 68 | 2072 approx. | 65 |
Table 1: Table represents two Phusion PCR reactions amplifying the plasmid pSB1C3 so that it can be ligated together with other Subtilin Immunity fragments using the 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.
Discussion
After gel electrophoresis the correct band was found with the original primers, but no band was found with the new primers. We will proceed to gel extraction of the correct band tomorrow.
Amplification of the parts for Subtilin Immunity BioBrick
Aim
The aim of this experiment is to amplify the four parts for the Subtilin Immunity BioBrick using Phusion PCR again but using the original primers that were ordered and hydrated. (Before this could be done, four of the primers, 1-T1_for, 2-T1_rev, 1-P1_for and 2-P2_rev, had to be re-hydrated.)
For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)
Materials and Protocol
Please refer to Phusion PCR for Phusion PCR protocol. (Please refer to DNA Re-hydration for DNA Re-hydration protocol.)
The details for the four PCR reactions are included in the table below:
| 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 | linear PSB1C3 (cut with HindIII) | P1V1 forward | P2V1 reverse | 53.3 (53) | 2046 + | 70 |
| 2 | Promoter and RBS (pVeg-SpoVG) | BioBrick Bba_K143053 | P1P1 forward | P2P2 reverse | 51.7 (51) | 139 + | 15 |
| 3 | spaIFEG Gene Cluster | B. subtilis ATCC 6633 | P1S1 forward | P2S1 reverse | 53.0 (53) | 2753 + | 110 |
| 4 | Double terminator | pSB1AK3 consisting BBa_B0014 | P1T1 forward | P2T1 reverse | 50.9 (51) | 116 + | 15 |
Table 2: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts will 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.
Results, Discussion and Conclusion
Gel electrophoresis and Gel extraction will be carried out if the bands are successful.
yneA
Single Digest
Aim
To do single digest of pGFPrrnB with Pst1 and Nhe1 to test if the enzymes are contaminated.
Materials and Protocol
Please refer to restriction digest and gel electrophoresis.
Results
Setting up Overnight Cultures
Aims
To prepare overnight cultures for minipreps of yneA, pGFPrrnB and pSB1C3, and also cultures for B. subtilis transformation.
Materials and Protocol
Please refer to growing an overnight culture.
Gibson cloning of the rocF BioBrick
Aim
The aim of this experiment is to assemble the fragments of the rocF BioBrick and clone it into pSB1C3 in a single step isothermal reaction. We performed five Gibson reactions to make ten transformations, for higher probability of the correct product. Last time we performed the Gibson reaction we had a high rate of incorrect products, resulting from re-circularisation of the plasmid pSB1C3.
Materials and Protocol
Please refer to: Gibson cloning and transformation of E. coli for materials required and the protocol for the reaction.
Result
The final result about the success of the Gibson reaction will be found on 2nd September, 2010.
   
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