Part:BBa_K2205000

J23100-RFP

BBa_K2205000 is a proposed composite BioBrick reporter that constitutively produces mRFP1. It possesses an mRFP1 coding sequence (E1010) after a strong Anderson promoter (J23100) and RBS (B0034). A double terminator (B0015) was added after the MRFP1 sequence. A BioBrick RFC10 prefix and suffix were added as well.

This part could be used as an alternative to BBa_J04450 for white-red colony selection as suggested by TU_Dresden22 team.
BBa_K2205000 can be used as a component of level 0 acceptor of RFC1000 assembly standard (BBa_K2205000 + pSB1C00). In this case, the insertion of a biological part in it will excise mRFP1, thus making transformant colonies white. Red colonies signal that the part has not been integrated.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 616
  Illegal AgeI site found at 728
  
• 1000
  COMPATIBLE WITH RFC[1000]

Part Documentation by TU_Dresden 2022 Team

Expression of mRFP1 under constitutive Anderson promoter

Measurement procedure

To measure the effect of the Anderson J23100 promoter on the RFP expression we used an analogous approach as described in the Interlab study.
This included:

1. overnight culture in LB of colonies of interest
2. dilution of overnight cultures down to 0.02 OD
3. pipetting of the diluted samples into 96-well plates followed by fluorescence and absorbance measurements.

We took continuous fluorescence measurement of growing DH5-alpha Escherichia coli in the temperature-controlled plate reader Tecan infinite200Pro with the following settings:

• 37°C with continuous shaking
• measurements conducted every 2 hrs over a 24 hrs period after the start of the experiment

Comparison to BBa_J04450

Firstly, we measured J4550 part with and without IPTG induction, and then compared it to BBa_K2205000 (mRFP1 with J23100 promoter) (see Fig. 1). We report that maximum level of fluorescence is not significant different in both parts (see Fig. 1B). However, the appearance of mRFP1 fluorescence in bacteria with BBa_K2205000 was 3 hours slower than in original J4550 part. To overcome this slow expression we additionally designed similar part but with turboRFP.

Figure 1: A. Lac promoter of BBa_J04450 part and mRFP1 reporter downstream in presence of inductor (IPTG) and in its absence. B. Fluorescence/absorbance 600nm of mRFP1 of the BBa_J04450 compared to BBa_K2205000 part with J23100 promoter over 24 hour incubation of bacterial culture.

Usage

BBa_K2205000 can mantain constant expression of mRFP1 in E. coli. It does not need an expensive IPTG for induction and mRFP1 expression is not prone to leakage. This makes this part a good candidate for colony selection.

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