Part:BBa_K2292005

Lactate detection

Our project aims to detect the risk of caries through three parameters: Competence stimulating peptide (CSP) concentration, lactate level, and pH value.

This construction is used for lactate quantization. We made use of the property of LldR operon. The LldR is transcription factor. Without lactate, it will bind on O1&O2 operator site, form a DNA loop, blocking the transcription. As lactate present, lactate will bind on LldR and open the loop, allowing the transcription to go on and produce GFP. GFP intensity is then detected and represent lactate level. This construction is cloned into E.coli.

Figure 1 Mechanism of lactate detection

O1 and O2 binding is added on two sides of promoter(J23118)

Sequence:

O1: AATTGGCCCTACCAATT O2: AATTGGCAGTGCCACTT

Figure 2 1 % Agarose, 100V. Check the size of assemble [pSB1A2 (2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR (2241 bp)].

Sample 1: [pSB1A2 (2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR (2241 bp)] digested with EcoRI and PstI

Sample 2: [pSB1A2 (2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR (2241 bp)] digested with EcoRI and PstI

Sample 3: [pSB1A2 (2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR (2241 bp)] digested with EcoRI and PstI

After finished assambling our parts, we did some function test, to find the relationship between GFP expression and lactate level.

Figure 3 Fluorescence of each lactate concentration depends on time.

We found that even though fluorescence in each concentration of lactate are floating, the spacing of each data are stable expect data 10 mM. It seems to be saturated when lactate concentration reaches 8 mM.

Figure 4 Fluorescence of different time depends on lactate level.

The interval 120 minute ≤ time ≤210 minute, the fluorescence of each concentration of lactate is similarity. Positive correlation exists between fluorescence and concentration of lactate on an interval 120 minute ≤ time ≤270 minute.

Figure 5 Combine Figure 3 and Figure 4 together.

We found that fluorescence level was high at the beginning. The general trend of fluorescence became pronounced and stable as time goes by.

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
  Illegal NheI site found at 993
  Illegal NheI site found at 1016
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 628
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1691