Part:BBa_K4115045

NS3-2-lac UV5 promoter-cscB-lacI-KanR-NS3-1

NS3-2-lac UV5 promoter-cscB-lacI-KanR-NS3-1
Function	permease
Use in	S.elongatus HL7942
RFC standard	None
Backbone	pUC57
Submitted by	ShanghaiTech_China

This composite part entered S.elongatus HL7942 by natural transformation with E. coli with the help of two homology arms NS3-1 (BBa_K3228025) and NS3-2 (BBa_K3971009), which originally come from S.elongatus HL7942. The core component of this composite part is cscB (BBa_K3971011), a membrane transporter that transports sucrose to the outside of the cell with the help of the H gradient. It can be expressed by a constitutive promoter J23101(BBa_J23101). We engineered cscB into HL7942 so that it can export sucrose to be used as the carbon source for E. coli.

Experiment Approach

1.Culture of S.elongatus HL7942
BG11 liquid medium(BG11 medium powder 1.7g, Milli-Q water 1L) and BG11 plate(BG11 medium powder 1.7g, Milli-Q water 1L, Agar powder 20g, NaS2O3·5H2O 4.8g). The liquid media is cultured in 30℃, 130rpm, 10W lamp tube, and solid media is cultured in 30℃, 8000lux.

2.Construction of the plasmid
We ligated the fragments together with Gibson Assembly and transferred them to the backbone of pUC57 and transformed it into E. coli.

3.Natural transformation
Take 1.5ml of bacterial fluid whose OD685 reaches 0.5 in the 1.5ml EP tube. Centrifuge to collect bacteria at 6000g for 10min. Discard the supernatant and use 750μl of non-resistant BG11 liquid medium for resuspension. Centrifuge to collect bacteria at 6000g for 10min. Repeat the front step. Discard the supernatant and use 250μl of non-resistant BG11 liquid medium for resuspension. Add plasmid solution into the tube. Make the concentration of DNA reach at least 1ng/μl. Wrap the 1.5ml EP tube with aluminium foil. Put it into the thermostatic dark shaker with 30℃ and 130rpm for 24 hours. After 24 hours, use a disposable spreader to evenly coat 100μl of the bacterial fluid on the resistant BG11 plate. Keep coating until no flowing liquid on the surface of the plate. Seal the plate with parafilm. Label and put the plate in the thermostatic light incubator. Culture for 5 days or until the single colony occurs. These single colonies are transformants.

4.The production of Sucrose by S.elongatus HL7942
Take 60ml of wild-type S.elongatus fluid whose OD685 reaches 0.5 in the centrifuge tube. Take 60ml of S.elongatus transformed with cscB gene fluid whose OD685 reaches 0.5 in the centrifuge tube.Centrifuge each group to collect bacteria at 6000g for 10min. Discard the supernatant and use the non-resistant BG11 liquid medium for resuspension of both groups. Centrifuge to collect bacteria at 6000g for 10min. Discard the supernatant. Use 60ml of non-resistant BG11 liquid medium for resuspension of wild type S.elongatus. Use 60ml of resistant BG11 liquid medium for resuspension of S.elongatus transformed with cscB gene.
Mix reagents according to the table below in the culture tubes:(C is control groups; E is experiment groups)

	Group C-1	Group C-2	Group C-3	Group C-4	Group E-1	Group E-2	Group E-3	Group E-4
Wild-type S. elongatus fluid (μl)	5000	4995	4500	4495	0	0	0	0
S. elongatus transformed with cscB gene fluid (μl)	0	0	0	0	5000	4995	4500	4495
1M IPTG solution (μl)	0	5	0	5	0	5	0	5
1M NaCl solution (μl)	0	0	500	500	0	0	500	500
Final volume of fluid (ml)	5	5	5	5	5	5	5	5
Number of parallel groups	3	3	3	3	3	3	3	3

Proof of function

We measured the concentration of sucrose and OD685 of S.elongatus HL7942 with cscB induced by NaCl and IPTG separately after 24h, 48h, and 72h. And then conclude the ratio of the concentration of surcose and OD685 of HL7942 as follow.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 507
  Illegal PstI site found at 4793
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 507
  Illegal PstI site found at 4793
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 507
  Illegal PstI site found at 4793
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 507
  Illegal PstI site found at 4793
  Illegal NgoMIV site found at 1678
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1722