Difference between revisions of "Part:BBa I714031:Experience"
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*For some data about the efficiency test of conjugation, please see the following link: [http://2007.igem.org/Peking_Hop-Count Data of Conjugation]
 
*For some data about the efficiency test of conjugation, please see the following link: [http://2007.igem.org/Peking_Hop-Count Data of Conjugation]
  
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An improvement of this part is using point mutation. The PCR system included BM1 plasmid backbone 2 μL, designed primer BM1 mut F 2 μL and BM1 mut R 2 μL, which can not be added together because they are complementary, PCR mixture 25 μL and ddH2O 19 μL. The single wrong base was corrected through designing the primer. After PCR, running gel electrophoresis and using gel image and gel recycle to obtain the PCR production. Transforming into cells to amplify plasmid and selecting monoclone through chloramphenicol medium. Target cells and sequencing results showed the right improvement.
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An improvement of this part is using point mutation. The PCR system is shown below:
 +
Plasmid_BBa_I714031    2 μL
 +
BM1_mut_F            2 μL
 +
BM1_mut_R            2 μL
 +
2×phanta Max Master Mix  25μL
 +
H2O                    19 μL
 +
Total                  50 μL
 +
The single wrong base was corrected through designing the primer. After PCR, running gel electrophoresis and using gel image and gel recycle to obtain the PCR production. Transforming into cells to amplify plasmid and selecting monoclone through chloramphenicol medium. Target cells and sequencing results showed the right improvement.

Revision as of 13:52, 10 October 2018

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I714031

Characterization of BBa_I714031

A [http://2009.igem.org/Team:TUDelft/ConjugationProtocol conjugation test] was done by the TUDelft iGEM 2009 team to characterize this part.

Donors: [http://www.cbs.knaw.nl/databases/nccb/search_bac_plas.aspx NCCBNr# 2350] cells with R751 and I13522+I714031-pSB4C5 (TRI+CAM)
Receivers: DH5 α cells with J23100 (AMP)
Transconjugants A: DH5 α cells with R751 and J23100 (TRI+AMP)
Transconjugants B: DH5 α cells with I13522+I714031-pSB4C5 and J23100 (CAM+AMP)

The following table shows the calculated conjugation efficiencies (raw data [http://2009.igem.org/Team:TUDelft/19_August_2009#Calin here]):

Set # Conjugation Efficiency for I13522+I714031-pSB4C5 Conjugation Efficiency for wild R751
1 0.00177 0.0141
2 0.00224 0.0476
3 n/a 0.0356


The suspected reason for the lower efficiency of I13522+I714031-pSB4C5 compared to R751 is the presence of the entry exclusion protein trbK. If a receiver cell gets I13522+I714031-pSB4C5, it can still receive R751 afterward. But if a receiver cell gets R751, it will not accept I13522+I714031-pSB4C5 afterward because trbK expression will block incoming transfers.

User Reviews

UNIQ417da5cc3de6ecf8-partinfo-00000000-QINU UNIQ417da5cc3de6ecf8-partinfo-00000001-QINU

  • For some data about the efficiency test of conjugation, please see the following link: [http://2007.igem.org/Peking_Hop-Count Data of Conjugation]

An improvement of this part is using point mutation. The PCR system is shown below: Plasmid_BBa_I714031 2 μL BM1_mut_F 2 μL BM1_mut_R 2 μL 2×phanta Max Master Mix 25μL H2O 19 μL Total 50 μL The single wrong base was corrected through designing the primer. After PCR, running gel electrophoresis and using gel image and gel recycle to obtain the PCR production. Transforming into cells to amplify plasmid and selecting monoclone through chloramphenicol medium. Target cells and sequencing results showed the right improvement.