DNA

Part:BBa_K4630100:Experience

Designed by: Zhejun Qin   Group: iGEM23_WHU-China   (2023-10-10)
Revision as of 20:07, 10 October 2023 by James Qin (Talk | contribs) (→‎Results)

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Applications of BBa_K4630100

This part is the key element we use in the proof-of-concept of the recorder. Also, it is used to measure the basic parameter of the recording event. The overall experiments we conduct on BBa_K4630100 includes:

  1. Verification of the self-targeting and self-recombination
  2. Exploration of the concentration and induction time reliance of the inducer
  3. Exclusion of the unexpected recombination

General Induction Protocol

  1. Pick a colony from the verified plate, cultivate in 5ml liquid LB media for 12 hours.
  2. Transfer 100µl bacteria solution to 3ml new liquid LB media. Cultivate to an OD600 of 0.3~0.5.
  3. Add L-Arabinose and IPTG to certain final concentration and induce for a certain time.
  4. Spread the diluted solution to plates with appropriate antibiotics, and cultivate for 12 hours.
  5. Pick the colonies and test the editing result.

Results

Strain Construction

We successfully co-transformed our working plasmid with pCas, the vector for Cas9 and Lambda Red, into Escherichia coli DH5α (fig 1).


Fig 1 Construction of the working strain
Colony PCR results of the recorder with barcode. The white hollow arrowheads indicate the target bands. Index 5, 6, 7, 8, 9, 10, 11, 12 were successfully co-transformed.

Pilot Experiment

We added a variable dosage of arabinose and IPTG close to the working concentration (tbl 1). Induction time for L-Arabinose is 22 hours and IPTG 5 hours. The sequencing result uncovered a successful editing (fig 2a). Large-scale screening revealed that condition 2 exhibited the highest efficiency, with efficiencies of 70%, 10%, and 38.1% for condition 2, 3, and 4, respectively (fig 2b). Non-induction controls substantiated that induction is the prerequisite for recording (fig 2c).

Table 1 The random test over inducers

Fig 2 The induction readout
(a) Sequencing result of the picked colonies in the conditions. “R” represents “parallel repeat”. Conditions 2, 3, and 4 exhibited positive knock-out signals.
(b) After induction, the target sequence is supposed to be truncated. The yellow reference line indicates the knock-out sample.
(c) Sequencing results of the non-induction controls. All of them remained intact. N = 24.

First Concentration Matrix

To better measure the property of the cassette, we maintained the induction time at the previous preferred levels (Condition 2, L-Arabinose and IPTG inducing 22hrs and 5hrs respectively), we carried out a concentration matrix test (tbl 2).

Table 2 Group design for the concentration matrix


It’s noted that the induction of arabinose inhibited the growth of bacteria strikingly (fig 3). Despite the missing data due to failure of sequencing, E6, E5, C2, B5 performed better (tbl 3, fig 4a, tbl 4), and the Lac promoter exhibited significant leakage expression (tbl 5, fig 4b). Furthermore, quality test based on electrophoresis provided parallel data for randomly picked groups, and the two data access showed a significant correlation, with paired t-test P = 0.7602, no significant difference (tbl 6, tbl 7, fig 5). Given the substantially larger amount of data from the electrophoresis test for E6 (N = 20) compared to sequencing (N = 4), we adjusted the knock-out rate of E6 to 60%.

Fig 3 Streaking plate of Group 1, after induction
(a) Bacteria without induction. The bacteria had grown to Area 3.
(b-d) Bacteria under 1, 4, 8 g/L L-Arabinose induction, respectively. The growth of the bacteria was limited to Areas 1 and 2. The bacteria in C1 had been contaminated.
(e) Assessment of the bacteria amount. The introduction of L-Arabinose influences the bacteria amount strikingly.

Fig 4 Results of the concentration matrix
(a) Electrophoresis result of several selected induction group. The yellow lines indicate the knock-out band.
(b) Heatmap of the concentration matrix based on sequencing data. The black block indicates no sequencing data is available. The L-Arabinose 0g/L row and IPTG 0 g/L column indicate the leakage expression of pBAD is quite low while that of pLac is quite high.
(c) Comparison in knock-out rate of IPTG 0 g/L column and the average of total. Though there is a slight increase along with the L- Arabinose concentration, the presence of L- Arabinose predominates, implying a high rate of pLac leakage.


Table 3 The total sample amount of each condition

Table 4 The groups with high knock-out rate

Table 5 Results of the no-IPTG-induction group

Table 6 The paired data of the electrophoresis and sequencing

Table 7 The correlation of the electrophoresis and sequencing result

Fig 5 The relation between data from sequencing and electrophoresis
(a) The correlation relationship of the two data. Pearson r = 0.9837, R squared = 0.9678, P = 0.0025(**).
(b) The normality test of the two data. Under Shapiro-Wilk test (N = 5), the P values for sequencing and electrophoresis are 0.9500 and 0.9364, respectively.
(c) The paired t test result.

Second Concentration Matrix

Figure. 1 DNA manipulating methods from the literatures

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