Difference between revisions of "Part:BBa K4183006"

 
 
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Construct lacZ and kfiC into the same expression frame.Promoter is Plac(R0010)
 
Construct lacZ and kfiC into the same expression frame.Promoter is Plac(R0010)
  
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===Usage and Biology===
 
===Usage and Biology===
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From the above experimental data, we obtained the engineered bacteria that can secrete lacZ into the extracellular and have some acid resistance. Therefore, we planned to combine the two, and thus developed and tested the first generation of engineered bacterium-lacMAN1.0 for lactos degradation.
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The results showed that the lacZ secretion capacity and acid tolerance of lacMan1.0 were reduced to different degrees (Figure 1). Only 13.33% of lacZ was secreted into the extracellular compartment, lower than the previous 21.93%; its growth remained slow at pH=4 but it barely survived at pH≤3. We think that this is caused by BBa_R0010 exhibiting insufficient promoter strength for an overly long expression frame.
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In addition, the induction of IPTG is extremely difficult in the intestine. Therefore, we planned to replace BBa_R0010 with the current promoter BBa_J23119, which is more strongly expressed, and deleted the original lacI element on the pET28a plasmid to reduce the replication pressure of bacteria.
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[[File:HS_China_engineering-8.png|900px|thumb|none|Figure1. Measurement of enzyme activity and acid resistance of LacMAN 1.0
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(A) The enzymatic activity of lacZ was determined in lacMAN1.0 in the bacteriophage as well as in the supernatant using the kit.
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(B) To determine the growth of lacMAN1.0 in acidic environment with different pH. ]]
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Latest revision as of 14:55, 11 October 2022


Construct lacZ and kfiC into the same expression frame.

Construct lacZ and kfiC into the same expression frame.Promoter is Plac(R0010)


Usage and Biology

From the above experimental data, we obtained the engineered bacteria that can secrete lacZ into the extracellular and have some acid resistance. Therefore, we planned to combine the two, and thus developed and tested the first generation of engineered bacterium-lacMAN1.0 for lactos degradation.

The results showed that the lacZ secretion capacity and acid tolerance of lacMan1.0 were reduced to different degrees (Figure 1). Only 13.33% of lacZ was secreted into the extracellular compartment, lower than the previous 21.93%; its growth remained slow at pH=4 but it barely survived at pH≤3. We think that this is caused by BBa_R0010 exhibiting insufficient promoter strength for an overly long expression frame.

In addition, the induction of IPTG is extremely difficult in the intestine. Therefore, we planned to replace BBa_R0010 with the current promoter BBa_J23119, which is more strongly expressed, and deleted the original lacI element on the pET28a plasmid to reduce the replication pressure of bacteria.

Figure1. Measurement of enzyme activity and acid resistance of LacMAN 1.0 (A) The enzymatic activity of lacZ was determined in lacMAN1.0 in the bacteriophage as well as in the supernatant using the kit. (B) To determine the growth of lacMAN1.0 in acidic environment with different pH.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3630
    Illegal PstI site found at 4762
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3630
    Illegal PstI site found at 4762
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3630
    Illegal BglII site found at 4212
    Illegal BamHI site found at 4565
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3630
    Illegal PstI site found at 4762
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3630
    Illegal PstI site found at 4762
  • 1000
    COMPATIBLE WITH RFC[1000]